U.S. patent application number 10/823785 was filed with the patent office on 2004-12-30 for staphylococcus aureus genes & polypeptides.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Bailey, Camella, Choi, Gil H..
Application Number | 20040265962 10/823785 |
Document ID | / |
Family ID | 34397131 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265962 |
Kind Code |
A1 |
Bailey, Camella ; et
al. |
December 30, 2004 |
Staphylococcus aureus genes & polypeptides
Abstract
The present invention relates to novel genes from S. aureus and
the polypeptides they encode. Also provided as are vectors, host
cells, antibodies and recombinant methods for producing the same.
The invention further relates to screening methods for identifying
agonists and antagonists of S. aureus polypeptide activity. The
invention additionally relates to diagnostic methods for detecting
Staphylococcus nucleic acids, polypeptides and antibodies in a
biological sample. The present invention further relates to novel
vaccines for the prevention or attenuation of infection by
Staphylococcus.
Inventors: |
Bailey, Camella;
(Washington, DC) ; Choi, Gil H.; (Rockville,
MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
34397131 |
Appl. No.: |
10/823785 |
Filed: |
April 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10823785 |
Apr 14, 2004 |
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10138701 |
May 6, 2002 |
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6753149 |
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10138701 |
May 6, 2002 |
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09512255 |
Feb 24, 2000 |
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6403337 |
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09512255 |
Feb 24, 2000 |
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08781986 |
Jan 3, 1997 |
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6737248 |
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09512255 |
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08956171 |
Oct 20, 1997 |
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6593114 |
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08956171 |
Oct 20, 1997 |
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08781986 |
Jan 3, 1997 |
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6737248 |
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09512255 |
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PCT/US99/19726 |
Aug 31, 1999 |
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60009861 |
Jan 5, 1996 |
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60009861 |
Jan 5, 1996 |
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60098964 |
Sep 1, 1998 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/320.1; 530/350; 536/23.7 |
Current CPC
Class: |
C12N 15/1093 20130101;
C07K 14/31 20130101; C12N 15/52 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/252.3; 435/320.1; 530/350; 536/023.7 |
International
Class: |
C07H 021/04; C12P
021/04; C12N 001/21; C07K 014/315 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding any one of the amino acid
sequences of the polypeptides shown in Table 1; (b) a nucleotide
sequence complementary to any one of the nucleotide sequences in
(a) (c) a nucleotide sequence at least 95% identical to any one of
the nucleotide sequences shown in Table 1; and (d) a nucleotide
sequence at least 95% identical to a nucleotide sequence
complementary to any one of the nucleotide sequences shown in Table
1.
2. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
identical to a nucleotide sequence in (a) or (b) of claim 1.
3. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which encodes an epitope-bearing portion of a
polypeptide in (a) of claim 1.
4. The isolated nucleic acid molecule of claim 3, wherein said
epitope-bearing portion of a polypeptide comprises an amino acid
sequence listed in Table 4.
5. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
6. A recombinant vector produced by the method of claim 5.
7. A host cell comprising the vector of claim 6.
8. A method of producing a polypeptide comprising: (a) growing the
host cell of claim 7 such that the protein is expressed by the
cell; and (b) recovering the expressed polypeptide.
9. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) a complete amino acid
sequence in Table 1; (b) a complete amino acid sequence of Table 1
except the N-terminal residue; (c) a fragment of a polypeptide of
Table 1 having biological activity; and (d) a fragment of a
polypeptide of Table 1 which binds to an antibody specific for a S.
aureus polypeptide.
10. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to an amino acid sequence of Table 1.
11. An isolated epitope-bearing polypeptide comprising an amino
acid sequence of Table 4.
12. An isolated antibody specific for the polypeptide of claim
9.
13. A cell which produces an antibody of claim 12.
14. A vaccine, comprising: (1) one or more S. aureus polypeptide(s)
of claim 9; and (2) a pharmaceutically acceptable diluent, carrier,
or excipient, wherein said polypeptide is present, in an amount
effective to elicit protective antibodies in an animal to a member
of the Staphylococcus genus.
15. A method of preventing or attenuating an infection caused by a
member of the Staphylococcus genus in an animal, comprising
administering to said animal a polypeptide of claim 9, wherein said
polypeptide is administered in an amount effective to prevent or
attenuate said infection.
16. A method of detecting Staphylococcus nucleic acids in a
biological sample comprising: (a) contacting the sample with one or
more nucleic acids of claim 1, under conditions such that
hybridization occurs; and (b) detecting hybridization of said
nucleic acids to the one or more Staphylococcus nucleic acid
sequences present in the biological sample.
17. A method of detecting Staphylococcus antibodies in a biological
sample obtained from an animal, comprising (a) contacting the
sample with a polypeptide of claim 9; and (b) detecting
antibody-antigen complexes.
18. A method of detecting a polypeptide of claim 9 comprising: (a)
obtaining a biological sample suspected of containing said
polypeptide; (c) contacting said sample with an antibody that binds
specifically to said polypeptide; and (c) determining the presence
or absence of said polypeptide in said biological sample.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/138,701, filed May 6, 2002, which is a divisional of U.S.
application Ser. No. 09/512,255, filed Feb. 24, 2000 (now U.S. Pat.
No. 6,403,337, issued Jun. 11, 2002), which is a
continuation-in-part of U.S. application Ser. No. 08/781,986, filed
Jan. 3, 1997, which claims benefit under 35 U.S.C. .sctn. 119(e) of
U.S. Provisional Application No. 60/009,861, filed Jan. 5, 1996;
U.S. application Ser. No. 09/512,255 is also a continuation-in-part
of U.S. patent application Ser. No. 08/956,171, filed Oct. 20, 1997
(now U.S. Pat. No. 6,593,114, issued Jul. 15, 2003), which is a
continuation-in-part of U.S. patent application Ser. No.
08/781,986, filed Jan. 3, 1997, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/009,861, filed Jan. 5, 1996; U.S. application Ser. No.
09/512,255 is also a continuation-in-part of International
Application No. PCT/US99/19726, filed Aug. 31, 1999, which claims
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application No. 60/098,964, filed Sep. 1, 1998. Each of the
above-listed priority applications is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel Staphylococcus aureus
genes (S. aureus) nucleic acids and polypeptides. Also provided are
vectors, host cells and recombinant methods for producing the same.
Further provided are diagnostic methods for detecting S. aureus
using probes, primers, and antibodies to the S. aureus nucleic
acids and polypeptides of the present invention. The invention
further relates to screening methods for identifying agonists and
antagonists of S. aureus polypeptide activity and to vaccines using
S. aureus nucleic acids and polypeptides.
BACKGROUND OF THE INVENTION
[0003] The genus Staphylococcus includes at least 20 distinct
species. (For a review see Novick, R. P., The Staphylococcus as a
Molecular Genetic System in MOLECULAR BIOLOGY OF THE STAPHYLOCOCCI,
1-37 (R. Novick, Ed., VCH Publishers, New York (1990)). Species
differ from one another by 80% or more, by hybridization kinetics,
whereas strains within a species are at least 90% identical by the
same measure.
[0004] The species S. aureus, a gram-positive, facultatively
aerobic, clump-forming cocci, is among the most important
etiological agents of bacterial infection in humans, as discussed
briefly below.
[0005] Human Health and S. aureus
[0006] Staphylococcus aureus is a ubiquitous pathogen. See, e.g.,
Mims et al., MEDICAL MICROBIOLOGY (Mosby-Year Book Europe Limited,
London, UK 1993). It is an etiological agent of a variety of
conditions, ranging in severity from mild to fatal. A few of the
more common conditions caused by S. aureus infection are burns,
cellulitis, eyelid infections, food poisoning, joint infections,
neonatal conjunctivitis, osteomyelitis, skin infections, surgical
wound infection, scalded skin syndrome and toxic shock syndrome,
some of which are described further below.
[0007] Burns: Burn wounds generally are sterile initially. However,
they generally compromise physical and immune barriers to
infection, cause loss of fluid and electrolytes and result in local
or general physiological dysfunction. After cooling, contact with
viable bacteria results in mixed colonization at the injury site.
Infection may be restricted to the non-viable debris on the burn
surface ("eschar"), it may progress into full skin infection and
invade viable tissue below the eschar and it may reach below the
skin, enter the lymphatic and blood circulation and develop into
septicemia. S. aureus is among the most important pathogens
typically found in burn wound infections. It can destroy
granulation tissue and produce severe septicemia.
[0008] Cellulitis: Cellulitis, an acute infection of the skin that
expands from a typically superficial origin to spread below the
cutaneous layer, most commonly is caused by S. aureus in
conjunction with S. pyrogenes. Cellulitis can lead to systemic
infection. In fact, cellulitis can be one aspect of synergistic
bacterial gangrene. This condition typically is caused by a mixture
of S. aureus and microaerophilic Streptococci. It causes necrosis
and treatment is limited to excision of the necrotic tissue. The
condition often is fatal.
[0009] Eyelid infections: S. aureus is the cause of styes and of
"sticky eye" in neonates, among other eye infections. Typically
such infections are limited to the surface of the eye, and may
occasionally penetrate the surface with more severe
consequences.
[0010] Food poisoning: Some strains of S. aureus produce one or
more of five serologically distinct, heat and acid stable
enterotoxins that are not destroyed by digestive process of the
stomach and small intestine (enterotoxins A-E). Ingestion of the
toxin, in sufficient quantities, typically results in severe
vomiting, but not diarrhea. The effect does not require viable
bacteria. Although the toxins are known, their mechanism of action
is not understood.
[0011] Joint infections: S. aureus infects bone joints causing
diseases such osteomyelitis. See, e.g., R. Cunningham et al.,
(1996) J. Med. Microbiol. 44:157-164.
[0012] Osteomyelitis: S. aureus is the most common causative agent
of haematogenous osteomyelitis. The disease tends to occur in
children and adolescents more than adults and it is associated with
non-penetrating injuries to bones. Infection typically occurs in
the long end of growing bone, hence its occurrence in physically
immature populations. Most often, infection is localized in the
vicinity of sprouting capillary loops adjacent to epiphysis growth
plates in the end of long, growing bones.
[0013] Skin infections: S. aureus is the most common pathogen of
such minor skin infections as abscesses and boils. Such infections
often are resolved by normal host response mechanisms, but they
also can develop into severe internal infections. Recurrent
infections of the nasal passages plague nasal carriers of S.
aureus.
[0014] Surgical Wound Infections: Surgical wounds often penetrate
far into the body. Infection of such wound thus poses a grave risk
to the patient. S. aureus is the most important causative agent of
infections in surgical wounds. S. aureus is unusually adept at
invading surgical wounds; sutured wounds can be infected by far
fewer S. aureus cells then are necessary to cause infection in
normal skin. Invasion of surgical wound can lead to severe S.
aureus septicemia. Invasion of the blood stream by S. aureus can
lead to seeding and infection of internal organs, particularly
heart valves and bone, causing systemic diseases, such as
endocarditis and osteomyelitis.
[0015] Scalded Skin Syndrome: S. aureus is responsible for "scalded
skin syndrome" (also called toxic epidermal necrosis, Ritter's
disease and Lyell's disease). This diseases occurs in older
children, typically in outbreaks caused by flowering of S. aureus
strains produce exfoliation(also called scalded skin syndrome
toxin). Although the bacteria initially may infect only a minor
lesion, the toxin destroys intercellular connections, spreads
epidermal layers and allows the infection to penetrate the outer
layer of the skin, producing the desquamation that typifies the
diseases. Shedding of the outer layer of skin generally reveals
normal skin below, but fluid lost in the process can produce severe
injury in young children if it is not treated properly.
[0016] Toxic Shock Syndrome: Toxic shock syndrome is caused by
strains of S. aureus that produce the so-called toxic shock
syndrome toxin. The disease can be caused by S. aureus infection at
any site, but it is too often erroneously viewed exclusively as a
disease solely of women who use tampons. The disease involves
toxemia and septicemia, and can be fatal.
[0017] Nocosomial Infections: In the 1984 National Nocosomial
Infection Surveillance Study ("NNIS") S. aureus was the most
prevalent agent of surgical wound infections in many hospital
services, including medicine, surgery, obstetrics, pediatrics and
newborns. Other Infections: Other types of infections, risk
factors, etc. involving S. aureus are discussed in: A. Trilla
(1995) J. Chemotherapy 3:37-43; F. Espersen (1995) J. Chemotherapy
3:11-17; D. E. Craven (1995) J. Chemotherapy 3:19-28; J. D. Breen
et al. (1995) Infect. Dis. Clin. North Am. 9(1): 11-24 (each
incorporated herein in their entireties).
[0018] Resistance to Drugs of S. aureus Strains
[0019] Prior to the introduction of penicillin the prognosis for
patients seriously infected with S. aureus was unfavorable.
Following the introduction of penicillin in the early 1940s even
the worst S. aureus infections generally could be treated
successfully. The emergence of penicillin-resistant strains of S.
aureus did not take long, however. Most strains of S. aureus
encountered in hospital infections today do not respond to
penicillin; although, fortunately, this is not the case for S.
aureus encountered in community infections.
[0020] It is well known now that penicillin-resistant strains of S.
aureus produce a lactamase which converts penicillin to
pencillinoic acid, and thereby destroys antibiotic activity.
Furthermore, the lactamase gene often is propagated episomally,
typically on a plasmid, and often is only one of several genes on
an episomal element that, together, confer multidrug
resistance.
[0021] Methicillins, introduced in the 1960s, largely overcame the
problem of penicillin resistance in S. aureus. These compounds
conserve the portions of penicillin responsible for antibiotic
activity and modify or alter other portions that make penicillin a
good substrate for inactivating lactamases. However, methicillin
resistance has emerged in S. aureus, along with resistance to many
other antibiotics effective against this organism, including
aminoglycosides, tetracycline, chloramphenicol, macrolides and
lincosamides. In fact, methicillin-resistant strains of S. aureus
generally are multiply drug resistant.
[0022] Methicillian-resistant S. aureus (MRSA) has become one of
the most important nosocomial pathogens worldwide and poses serious
infection control problems. Today, many strains are multiresistant
against virtually all antibiotics with the exception of
vancomycin-type glycopeptide antibiotics.
[0023] Recent reports that transfer of vancomycin resistance genes
from enterococci to S. aureus has been observed in the laboratory
sustain the fear that MRSA might become resistant against
vancomycin, too, a situation generally considered to result in a
public health disaster. MRSA owe their resistance against virtually
all .beta.-lactam antibiotics to the expression of an extra
penicillin binding protein (PBP) 2a, encoded by the mecA gene. This
additional very low affinity pbp, which is found exclusively in
resistant strains, appears to be the only pbp still functioning in
cell wall peptidoglycan synthesis at .beta.-lactam concentrations
high enough to saturate the normal set of S. aureus pbp 1-4. In
1983 it was shown by insertion mutagenesis using transposon Tn551
that several additional genes independent of mecA are needed to
sustain the high level of methicillin resistance of MRSA.
Interruption of these genes did not influence the resistance level
by interfering with PBP2a expression, and were therefore called fem
(factor essential for expression of methicillin resistance) or aux
(auxiliary genes).
[0024] Six fem genes (femA-through F) have been described and the
minimal number of additional aux genes has been estimated to be
more than 10. Interference with fem A and fem B results in a strong
reduction of methicillin resistance, back to sensitivity of strains
without PBP2a. The fem genes are involved in specific steps of cell
wall synthesis. Consequently, inactivation of fem encoded factors
induce .beta.-lactam hypersensitivity in already sensitive strains.
Both femA and fem B have been shown to be involved in peptidoglycan
pentaglycine interpeptide bridge formation. FemA is responsible for
the formation of glycines 2 and 3, and FemB is responsible for
formation of glycines 4 and 5. S. aureus may be involved in the
formation of a monoglycine muropeptide precursors. FemC-F influence
amidation of the iso-D-glutamic acid residue of the peptidoglycan
stem peptide, formation of a minor muropeptide with L-alanine
instead of glycine at position 1 of the interpeptide bridge,
perform a yet unknown function, or are involved in an early step of
peptidoglycan precursors biosynthesis (addition of L-lysine),
respectively.
SUMMARY OF THE INVENTION
[0025] The present invention provides isolated S. aureus
polynucleotides and polypeptides shown in Table 1 and SEQ ID NO: 1
through SEQ ID NO:61. One aspect of the invention provides isolated
nucleic acid molecules comprising or alternatively consisting of
polynucleotides having a nucleotide sequence selected from the
group consisting of: (a) a nucleotide sequence shown in Table 1;
(b) a nucleotide sequence encoding any of the amino acid sequences
of the polypeptides shown in Table 1; and (c) a nucleotide sequence
complementary to any of the nucleotide sequences in (a) or (b). The
invention further provides for fragments of the nucleic acid
molecules of (a), (b) & (c) above.
[0026] Further embodiments of the invention include isolated
nucleic acid molecules that comprise, or alternatively consist of,
a polynucleotide having a nucleotide sequence at least 90%
identical, and more preferably at least 95%, 96%, 97%, 98% or 99%
identical, to any of the nucleotide sequences in (a), (b) or (c)
above, or a polynucleotide which hybridizes under stringent
hybridization conditions to a polynucleotide in (a), (b) or (c)
above. Additional nucleic acid embodiments of the invention relate
to isolated nucleic acid molecules comprising polynucleotides which
encode the amino acid sequences of epitope-bearing portions of a S.
aureus polypeptide having an amino acid sequence in Table 1, and
including but not limited to those epitope-bearing portions shown
in Table 4.
[0027] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells. The
present invention further relates to the use of these vectors in
the production of S. aureus polypeptides or peptides by recombinant
techniques.
[0028] The invention further provides isolated S. aureus
polypeptides having an amino acid sequence selected from the group
consisting of an amino acid sequence of any of the polypeptides
described in Table 1 or fragments thereof.
[0029] The polypeptides of the present invention also include
polypeptides having an amino acid sequence with at least 70%
similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% similarity to those described in Table 1, as
well as polypeptides having an amino acid sequence at least 70%
identical, more preferably at least 75% identical, and still more
preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
an amino acid sequence of any of the polypeptides described in
Table 1 or fragments thereof. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0030] The present invention further provides a vaccine, preferably
a multi-component vaccine comprising one or more of the S. aureus
polynucleotides or polypeptides described in Table 1, or fragments
thereof, together with a pharmaceutically acceptable diluent,
carrier, or excipient, wherein the S. aureus polypeptide(s) are
present in an amount effective to elicit an immune response to
members of the Staphylococcus genus, or at least S. aureus, in an
animal. The S. aureus polypeptides of the present invention may
further be combined with one or more immunogens of one or more
other staphylococcal or non-staphylococcal organisms to produce a
multi-component vaccine intended to elicit an immunological
response against members of the Staphylococcus genus and,
optionally, one or more non-staphylococcal organisms.
[0031] The vaccines of the present invention can be administered in
a DNA form, e.g., "naked" DNA, wherein the DNA encodes one or more
staphylococcal polypeptides and, optionally, one or more
polypeptides of a non-staphylococcal organism. The DNA encoding one
or more polypeptides may be constructed such that these
polypeptides are expressed as fusion proteins.
[0032] The vaccines of the present invention may also be
administered as a component of a genetically engineered organism or
host cell. Thus, a genetically engineered organism or host cell
which expresses one or more S. aureus polypeptides may be
administered to an animal. For example, such a genetically
engineered organism or host cell may contain one or more S. aureus
polypeptides of the present invention intracellularly, on its cell
surface, or in its periplasmic space. Further, such a genetically
engineered organism or host cell may secrete one or more S. aureus
polypeptides. The vaccines of the present invention may also be
co-administered to an animal with an immune system modulator (e.g.,
CD86 and GM-CSF).
[0033] The invention also provides a method of inducing an
immunological response in an animal to one or more members of the
Staphylococcus genus, preferably one or more isolates of the S.
aureus species, comprising administering to the animal a vaccine as
described above.
[0034] The invention further provides a method of inducing a
protective immune response in an animal, sufficient to prevent,
attenuate, or control an infection by members of the Staphylococcus
genus, preferably at least S. aureus species, comprising
administering to the animal a composition comprising one or more of
the polynucleotides or polypeptides described in Table 1, or
fragments thereof (e.g., including, but not limited to, fragments
which comprise the epitopes shown in Table 4). Further, these
polypeptides, or fragments thereof, may be conjugated to another
immunogen and/or administered in admixture with an adjuvant.
[0035] The invention further relates to antibodies elicited in an
animal by the administration of one or more S. aureus polypeptides
of the present invention and to methods for producing such
antibodies and fragments thereof. The invention further relates to
recombinant antibodies and fragments thereof and to methods for
producing such antibodies and fragments thereof.
[0036] The invention also provides diagnostic methods for detecting
the expression of the polynucleotides and polypeptides of Table 1
by members of the Staphylococcus genus in a biological or
environmental sample. One such method involves assaying for the
expression of a polynucleotide encoding S. aureus polypeptides in a
sample from an animal. This expression may be assayed either
directly (e.g., by assaying polypeptide levels using antibodies
elicited in response to amino acid sequences described in Table 1
or fragments thereof) or indirectly (e.g., by assaying for
antibodies having specificity for amino acid sequences described in
Table 1 or fragments thereof). The expression of polynucleotides
can also be assayed by detecting the nucleic acids of Table 1. An
example of such a method involves the use of the polymerase chain
reaction (PCR) to amplify and detect Staphylococcus nucleic acid
sequences in a biological or environmental sample.
[0037] The invention also includes a kit for analyzing samples for
the presence of members of the Staphylococcus genus in a biological
or environmental sample. In a general embodiment, the kit includes
at least one polynucleotide probe containing a nucleotide sequence
that will specifically hybridize with a S. aureus nucleic acid
molecule of Table 1 and a suitable container. In a specific
embodiment, the kit includes two polynucleotide probes defining an
internal region of the S. aureus nucleic acid molecule of Table 1,
where each probe has one strand containing a 31'mer-end internal to
the region. In a further embodiment, the probes may be useful as
primers for polymerase chain reaction amplification.
[0038] The present invention also relates to nucleic acid probes
having all or part of a nucleotide sequence described in Table 1
which are capable of hybridizing under stringent conditions to
Staphylococcus nucleic acids. The invention further relates to a
method of detecting one or more Staphylococcus nucleic acids in a
biological sample obtained from an animal, said one or more nucleic
acids encoding Staphylococcus polypeptides, comprising: (a)
contacting the sample with one or more of the above-described
nucleic acid probes, under conditions such that hybridization
occurs, and (b) detecting hybridization of said one or more probes
to the Staphylococcus nucleic acid present in the biological
sample.
[0039] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains S. aureus polypeptides or
polynucleotides of the invention. As indicated, biological samples
include body fluids (such as semen, lymph, sera, plasma, urine,
synovial fluid and spinal fluid) which contain the S. aureus
polypeptides or polynucleotides of the invention, and tissue
sources found to contain the expressed S. aureus polypeptides shown
in Table 1. Methods for obtaining tissue biopsies and body fluids
from mammals are well known in the art. Where the biological sample
is to include mRNA, a tissue biopsy is the preferred source.
[0040] The method(s) provided above may preferrably be applied in a
diagnostic method and/or kits in which S. aureus polynucleotides
and/or polypeptides of the invention are attached to a solid
support. In one exemplary method, the support may be a "gene chip"
or a "biological chip" as described in U.S. Pat. Nos. 5,837,832,
5,874,219, and 5,856,174. Further, such a gene chip with S. aureus
polynucleotides of Table 1 attached may be used to diagnose S.
aureus infection in a mammal, preferably a human. The US Patents
referenced above are incorporated herein by reference in their
entirety.
DETAILED DESCRIPTION
[0041] The present invention relates to recombinant antigenic S.
aureus polypeptides and fragments thereof. The invention also
relates to methods for using these polypeptides to produce
immunological responses and to confer immunological protection to
disease caused by members of the genus Staphylococcus. The
invention further relates to nucleic acid sequences which encode
antigenic S. aureus polypeptides and to methods for detecting
Staphylococcus nucleic acids and polypeptides in biological
samples. The invention also relates to Staphylococcus specific
antibodies and methods for detecting such antibodies produced in a
host animal.
[0042] Definitions
[0043] The following definitions are provided to clarify the
subject matter which the inventors consider to be the present
invention.
[0044] As used herein, the phrase "pathogenic agent" means an agent
which causes a disease state or affliction in an animal. Included
within this definition, for examples, are bacteria, protozoans,
fungi, viruses and metazoan parasites which either produce a
disease state or render an animal infected with such an organism
susceptible to a disease state (e.g., a secondary infection).
Further included are species and strains of the genus
Staphylococcus which produce disease states in animals.
[0045] As used herein, the term "organism" means any living
biological system, including viruses, regardless of whether it is a
pathogenic agent.
[0046] As used herein, the term "Staphylococcus" means any species
or strain of bacteria which is members of the genus Staphylococcus
regardless of whether they are known pathogenic agents.
[0047] As used herein, the phrase "one or more S. aureus
polypeptides of the present invention" means the amino acid
sequence of one or more of the S. aureus polypeptides disclosed in
Table 1. These polypeptides may be expressed as fusion proteins
wherein the S. aureus polypeptides of the present invention are
linked to additional amino acid sequences which may be of
Staphylococcal or non-Staphylococcal origin. This phrase further
includes fragments of the S. aureus polypeptides of the present
invention.
[0048] As used herein, the phrase "full-length amino acid sequence"
and "full-length polypeptide" refer to an amino acid sequence or
polypeptide encoded by a full-length open reading frame (ORF). For
purposes of the present invention, polynucleotide ORFs in Table 1
are defined by the corresponding polypeptide sequences of Table 1
encoded by said polynucleotide. Therefore, a polynucleotide ORF is
defined at the 5' end by the first base coding for the initiation
codon of the corresponding polypeptide sequence of Table 1 and is
defined at the 3' end by the last base of the last codon of said
polypeptide sequence. As is well known in the art, initiation
codons for bacterial species may include, but are not limited to,
those encoding Methionine, Valine, or Leucine. As discussed below
for polynucleotide fragments, the ORFs of the present invention may
be claimed by a 5' and 3' position of a polynucleotide sequence of
the present invention wherein the first base of said sequence is
position 1.
[0049] As used herein, the phrase "truncated amino acid sequence"
and "truncated polypeptide" refer to a sub-sequence of a
full-length amino acid sequence or polypeptide. Several criteria
may also be used to define the truncated amino acid sequence or
polypeptide. For example, a truncated polypeptide may be defined as
a mature polypeptide (e.g., a polypeptide which lacks a leader
sequence). A truncated polypeptide may also be defined as an amino
acid sequence which is a portion of a longer sequence that has been
selected for ease of expression in a heterologous system but
retains regions which render the polypeptide useful for use in
vaccines (e.g., antigenic regions which are expected to elicit a
protective immune response).
[0050] Additional definitions are provided throughout the
specification.
[0051] Explanation of Table 1
[0052] Table 1 lists the full length S. aureus polynucleotide and
polypeptide sequences of the present invention. Each polynucleotide
and polypeptide sequence is proceeded by a gene identifier. Each
polynucleotide sequence is followed by at least one polypeptide
sequence encoded by said polynucleotide. For some of the sequences
of Table 1, a known biological activity and the name of the homolog
with similar activity is listed after the gene sequence
identifier.
[0053] Explanation of Table 2
[0054] Table 2 lists accession numbers for the closest matching
sequences between the polypeptides of the present invention and
those available through GenBank and GeneSeq databases. These
reference numbers are the database entry numbers commonly used by
those of skill in the art, who will be familar with their
denominations. The descriptions of the nomenclature for GenBank are
available from the National Center for Biotechnology Information.
Column 1 lists the polynucleotide sequence of the present
invention. Column 2 lists the accession number of a "match" gene
sequence in GenBank or GeneSeq databases. Column 3 lists the
description of the "match" gene sequence. Columns 4 and 5 are the
high score and smallest sum probability, respectively, calculated
by BLAST. Polypeptides of the present invention that do not share
significant identity/similarity with any polypeptide sequences of
GenBank and GeneSeq are not represented in Table 2. Polypeptides of
the present invention that share significant identity/similarity
with more than one of the polypeptides of GenBank and GeneSeq may
be represented more than once.
[0055] Explanation of Table 3.
[0056] The S. aureus polypeptides of the present invention may
include one or more conservative amino acid substitutions from
natural mutations or human manipulation as indicated in Table 3.
Changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the
folding or activity of the protein. Residues from the following
groups, as indicated in Table 3, may be substituted for one
another: Aromatic, Hydrophobic, Polar, Basic, Acidic, and
Small,
[0057] Explanation of Table 4
[0058] Table 4 lists residues comprising antigenic epitopes of
antigenic epitope-bearing fragments present in each of the S.
aureus polypeptides described in Table 1 as predicted by the
inventors using the algorithm of Jameson and Wolf, (1988) Comp.
Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was
performed using the computer program PROTEAN (Version 3.11 for the
Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison,
Wis.). S. aureus polypeptides shown in Table 1 may possess one or
more antigenic epitopes comprising residues described in Table 4.
It will be appreciated that depending on the analytical criteria
used to predict antigenic determinants, the exact address of the
determinant may vary slightly. The residues and locations shown and
described in Table 4 correspond to the amino acid sequences for
each polypeptide sequence shown in Table 1 and in the Sequence
Listing. Polypeptides of the present invention that do not have
antigenic epitopes recognized by the Jameson-Wolf algorithm are not
represented in Table 2.
[0059] Nucleic Acid Molecules
[0060] Sequenced S. aureus genomic DNA was obtained from the S.
aureus strain ISP3. S. aureus strain ISP3, has been deposited at
the American Type Culture Collection, as a convenience to those of
skill in the art. The S. aureus strain ISP3 was deposited on 7 Apr.
1998 at the ATCC, 10801 University Blvd. Manassas, Va. 20110-2209,
and given accession number 202108. As discussed elsewhere herein,
polynucleotides of the present invention readily may be obtained by
routine application of well known and standard procedures for
cloning and sequencing DNA. A wide variety of S. aureus strains can
be used to prepare S. aureus genomic DNA for cloning and for
obtaining polynucleotides and polypeptides of the present
invention. A wide variety of S. aureus strains are available to the
public from recognized depository institutions, such as the
American Type Culture Collection (ATCC). It is recognized that
minor variations is the nucleic acid and amino acid sequence may be
expected from S. aureus strain to strain. The present invention
provides for genes, including both polynucleotides and
polypeptides, of the present invention from all the S. aureus
strains.
[0061] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90%
identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced
DNA molecule. The actual sequence can be more precisely determined
by other approaches including manual DNA sequencing methods well
known in the art. By "nucleotide sequence" of a nucleic acid
molecule or polynucleotide is intended to mean either a DNA or RNA
sequence. Using the information provided herein, such as the
nucleotide sequence in Table 1, a nucleic acid molecule of the
present invention encoding a S. aureus polypeptide may be obtained
using standard cloning and screening procedures, such as those for
cloning DNAs using genomic DNA as starting material. See, e.g.,
Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring
Harbor, N.Y. 2nd ed. 1989); Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (John Wiley and Sons, N. Y. 1989). Illustrative
of the invention, the nucleic acid molecule described in Table 1
was discovered in a DNA library derived from a S. aureus ISP3
genomic DNA.
1TABLE 1 Nucleotide and Amino Acid Sequences of S. aureus Genes.
>HGS001, SEQ ID NO: 1, fabH, 3-oxoacyl-acyl-carrier protein
synthase ATTAACTAGTCAATATTCCTACCTCTGACTTGAGTTTAAAAAGTAATCTA
TGTTAAATTAATACCTGGTATTAAAAATTTTATTAAGAAGGTGTTCAACT
ATGAACGTGGGTATTAAAGGTTTTGGTGCATATGCGCCAGAAAAGATTAT
TGACAATGCCTATTTTGAGCAATTTTTAGATACATCTGATGAATGGATTT
CTAAGATGACTGGAATTAAAGAAAGACATTGGGCAGATGATGATCAAGAT
ACTTCAGATTTAGCATATGAAGCAAGTTTAAAAGCAATCGCTGACGCTGG
TATTCAGCCCGAAGATATAGATATGATAATTGTTGCCACAGCAaCTGGaG
ATATGCCATTTCCAACTGTCGCAAATATGTTGCAAGAACGTTTAGGGACG
GGCAAAGTTGCCTCTATGGATCAACTTGCAGCATGTTCTGGATTTATGTA
TTCAATGATTACAGCTAAACAATATGTTCAATCTGGAGATTATCATAACA
TTTTAGTTGTCGGTGCAGATAAATTATCTAAAATAACAGATTTAACTGAC
CGTTCTACTGCAGTTCTATTTGGAGATGGTGCAGGTGCGGTTATCATCGG
TGAAGTTTCAGATGGCAGAGGTATTATAAGTTATGAAATGGGTTCTGATG
GCACAGGTGGTAACATTTATATTTAGATAAAGATACTGGTAAACTGAAAA
TGAATGGTCGAGAAGTATTTAAATTTGCTGTTAGAATTATGGGTGATGCA
TCAACACGTGTAGTTGAAAAAGCGAATTTAACATCAGATGATATAGATTT
ATTTATTCCTCATCAAGCTAATATTAGAATTATGGAATCAGCTAGAGAAC
GCTTAGGTATTTCAAAAGACAAAATGAGTGTTTCTGTAAATAAATATGGA
AATACTTCAGCTGCGTCAATACCTTTAAGTATCGATCAAGAATTAAAAAA
TGGTAAAATCAAAGATGATGATACAATTGTTCTTGTCGGATTCGGTGGCG
GCCTAACTTGGGGCGCAATGACAATAAAATGGGGAAAATAGGAGGATAAC
GAATGAGTCAAAATAAAAGAGTAGTTATTACAGGTATGGGA >HGS001, SEQ ID NO: 2,
FabH, 3-oxoacyl-acyl-carrier protein synthase
MINVGIKGFGAYAPEKIIDNAYFEQFLDTSDEWISKMTGIKERHWADDDQ
DTSDLAYEASLKAIADAGIQPEDIDMIIVATATGDMPFPTVANMLQERLG
TGKVASMDQLAACSGFMYSMITAICQYVQSGDYHNILVVGADKLSKITDL
TDRSTAVLFGDGAGAVIIGEVSDGRGIISYEMGSDGTGGKHLYLDKDTGK
LKMNGREVFKFAVRIMGDASTRVVEKANLTSDDIDLFIPHQANIRIMESA
RERLGISKDKMSVSVNKYGNTSAASIPLSIDQELKNGKIKDDDTIVLVGF GGGLTWGAMTIKWGK
>HGS002, SEQ ID NO: 3, murB, UDP-N-acetylenolpyruvoylglucosamine
reductase ATACTAATTCTAATACTTTCTTTTCAATTTTCGCAAATGAATTTTAAAAT
TGGTATAATACTATATGATATTAAAGACATGAGAAAGGATGTACTGAGAA
GTGATAAATAAAGACATCTATCAAGCTTTACAACAACTTATCCCAAATGA
AAAAATTAAAGTTGATGAACCTTTAAAACGATACACTTATACTAAAACAG
GTGGTAATGCCGACTTTTACATTACCCCTACTAAAAATGAAGAAGTACAA
GCAGTTGTTAAATATGCCTATCAAAATGAGATTCCTGTTACATATTTAGG
AAATGGCTCAAATATTATTATCCGTGAAGGTGGTATTCGCGGTATTGTAA
TTAGTTTATTATCACTAGATCATATCGAAGTATCTGATGATGCGATAATA
GCCGGTAGCGGCGCTGCAATTATTGATGTCTCACGTGTTGCTCGTGATTA
CGCACTTACTGGCCTTGAATTTGCATGTGGTATTCCAGGTTCAATTGGTG
GTGCAGTGTATATGAATGCTGGCGCTTATGGTGGCGAAGTTAAAGATTGT
ATAGACTATGCGCTTTGCGTAAACGAACAAGGCTCGTTAATTAAACTTAC
AACAAAAGAATTAGAGTTAGATTATCGTAATAGCATTATTCAAAAAGAAC
ACTTAGTTGTATTAGAAGCTGCATTTACTTTAGCTCCTGGTAAAATGACT
GAAATACAAGCTAAAATGGATGATTTAACAGAACGTAGAGAATCTAAACA
ACCTTTAGAGTATCCTTCATGTGGTAGTGTATTCCAAAGACCGCCTGGTC
ATTTTGCAGGTAAATTGATACAAGATTCTAATTTGCAAGGTCACCGTATT
GGCGGCGTTGAAGTTTCAACCAAACACGCTGGTTTTATGGTAAATGTAGA
CAATGGAACTGCTACAGATTATGAAAACCTTATTCATTATGTACAAAAGA
CCGTCAAAGAAAAATTTGGCATTGAATTAAATCGTGAAGTTCGCATTATT
GGTGAACATCCAAAGGAATCGTAAGTTAAGGAGCTTTGTCTATGCCTAAA
GTTTATGGTTCATTAATCGATACT >HGS002, SEQ ID NO: 4, MurB,
UDP-N-acetylenolpyruvoylglucosamine reductase
VINXDIYQALQQLIPNEKIKVDEPLKRYTYTKTGGNADFYITPTKNEEVQ
AVVKYAYQNEIPVTYLGNGSNIIIREGGIRGIVISLLSLDHIEVSDDAII
AGSGAAIIDVSRVARDYALTGLEFACGIPGSIGGAVYMNAGAYGGEVKDC
IDYALCVNEQGSLIKLTTKELELDYRNSIIQKEHLVVLEAAFTLAPGKMT
EIQAKMDDLTERRESKQPLEYPSCGSVFQRPPGHFAGKLIQDSNLQGERI
GGVEVSTKHAGFMVNVDNGTATDYENLIHYVQKTVKEKFGIELNREVRII GEHPKES
>HGS003, SEQ ID NO: 5, fabI, enoyl- acyl-carrier protein
reductase AATAGTGTTAAAATGTATTGACGAATAAAAAGTTA- GTTAAAACTGGGATT
AGATATTCTATCCGTTAAATTAATTATTATAAGGAGTTATCT- TACATGTT
AAATCTTGAAAACAAAACATATGTCATCATGGGAATCGCTAATAAGCGT- A
GTATTGCTTTTGGTGTCGCTAAAGTTTTAGATCAATTAGGTGCTAAATTA
GTATTTACTTACCGTAAAGAACGTAGCCGTAAAGAGCTTGAAAAATTATT
AGAACAATTAAATCAACCAGAAGCGCACTTATATCAAATTGATGTTCAAA
GCGATGAAGAGGTTATTAATGGTTTTGAGCAAATTGGTAAAGATGTTGGC
AATATTGATGGTGTATATCATTCAATCGCATTTGCTAATATGGAAGACTT
ACGCGGACGCTTTTCTGAAACTTCACGTGAAGGCTTCTTGTTAGCTCAAG
ACATTAGTTCTTACTCATTAACAATTGTGGCTCATGAAGCTAAAAAATTA
ATGCCAGAAGGTGGTAGCATTGTTGCAACAACATATTTAGGTGGCGAATT
CGCAGTTCAAAACTATAATGTGATGGGTGTTGCTAAAGCGAGCTTAGAAG
CAAATGTTAAATATTTAGCATTAGACTTAGGTCCAGATAATATTCGCGTT
AATGCAATTTCAGCTAGTCCAATCCGTACATTAAGTGCAAAAGGTGTGGG
TGGTTTCAATACAATTCTTAAAGAAATCGAAGAGCGTGCACCTTTAAAAC
GTAATGTTGATCAAGTAGAAGTAGGTAAAACTGCGGCTTACTTATTAAGT
GATTTATCAAGTGGCGTTACAGGTGAAAATATTCATGTAGATAGCGGATT
CCACGCAATTAAATAATATCATTCAACAGCTTTGTTCACGTTATTATATA TGTGAGCAAAGCTTTT
>HGS003, SEQ ID NO: 6, FabI, enoyl- acyl-carrier protein
reductase MLNLENKTYVIMGIANKRSIAFGVAKVL- DQLGAKLVFTYRXERSRXELEK
LLEQLNQPEAHLYQIDVQSDEEVINGFEQIGKDVG- NIDGVYHSIAFANME
DLRGRFSETSREGFLLAQDISSYSLTIVAHEAKKLMPEGGSI- VATTYLGG
EFAVQNYNVMGVAKASLEANVKYLALDLGPDNIRVNAISASPIRTLSAK- G
VGGFNTILKEIEERAPLKRNVDQVEVGKTAAYLLSDLSSGVTGENIHVDS GFHAIK
>HGS004, SEQ ID NO: 7, murA, UDP-N-acetylglucosamine
1-carboxyvinyltransferase
TAAAATAATTTTAAAATAGGGAAATGTAAAGTAATAGGAGTTCTAAGTGG
AGGATTTACGATGGATAAAATAGTAATCAAAGGTGGAAATAAATTAACGG
GTGAAGTTAAAGTAGAAGGTGCTAAAAATGCAGTATTACCAATATTGACA
GCATCTTTATTAGCTTCTGATAAACCGAGCAAATTAGTTAATGTTCCAGC
TTTAAGTGATGTAGAAACAATAAATAATGTATTAACAACTTTAAATGCTG
ACGTTACATACAAAAAGGACGAAAATGCTGTTGTCGTTGATGCAACAAAG
ACTCTAAATGAAGAGGCACCATATGAATATGTTAGTAAAATGCGTGCAAG
TATTTTAGTTATGGGACCTCTTTTAGCAAGACTAGGACATGCTATTGTTG
CATTGCCTGGTGGTTGTGCATTGGAAGTAGACCGATTGAGCAACACATTA
AAGGTTTTGAAGCTTTAGGCGCAGAAATTCATCTTGAAAATGGTAATATT
TATGCTAATGCTAAAGATGGATTAAAAGGTACATCAATTCATTTAGATTT
TCCAAGTGTAGGAGCAACACAAAATATTATTATGGCAGCATCATTAGCTA
AGGGTAAGACTTTAATTGAAAATGCAGCTAAAGAACCTGAAATTGTCGAT
TTAGCAAACTACATTAATGAAATGGGTGGTAGAATTACTGGTGCTGGTAC
AGACACAATTACAATCAATGGTGTAGAATCATTACATGGTGTAGAACATG
CTATCATTCCAGATAGATTGAAGCAGGCACATTACTAATCGCTGGTGCTA
TAACGCGTGGTGATATTTTTGTACGTGGTGCAATCAAAGAACATATGGCG
AGTTTAGTCTATAAACTAGAAGAAATGGGCGTTGAATTGGACTATCAAGA
AGATGGTATTCGTGTACGTGCTGAAGGGGAATTACAACCTGTAGACATCA
AAACTCTACCACATCCTGGATTCCCGACTGATATGCAATCACAAATGATG
GCATTGTTATTAACGGCAAATGGTCATAAAGTCGTAACCGAAACTGTTTT
TGAAAACCGTTTTATGCATGTTGCAGAGTTCAAACGTATGAATGCTAATA
TCAATGTAGAAGGTCGTAGTGCTAAACTTGAAGGTAAAAGTCAATTGCAA
GGTGCACAAGTTAAAGCGACTGATTTAAGAGCAGCAGCCGCCTTAATTTT
AGCTGGATTAGTTGCTGATGGTAAAACAAGCGTTACTGAATTAACGCACC
TAGATAGAGGCTATGTTGACTTACACGGTAAATTGAAGCAATTAGGTGCA
GACATTGAACGTATTAACGATTAATTCAGTAAATTAATATAATGGAGGAT
TTCAACCATGGAAACAATTTTTGA >HGS004, SEQ ID NO: 8, MurA,
UDP-N-acetylglucosamine 1-carboxyvinyltransferase
MDKIVIKGGNKLTGEVKVEGAKNAVLPILTASLLASDKPSKLVNVPALSD
VETINNVLTTLNADVTYKKDENAVVVDATKTLNEEAPYEYVSKMRASILV
MGPLLARLGHAIVALPGGCAIGSRPIEQHIKGFEALGAEIHLENGNIYAN
AKDGLKGTSIHLDFPSVGATQNIIMAASLAKGKTLIENAAXEPEIVDLAN
YINEMGGRITGAGTDTITINGVESLHGVEHAIIPDRIEAGTLLIAGAITR
GDIFVRGAIKEHMASLVYKLEEMGVELDYQEDGIRVRAEGELQPVDIKTL
PHPGFPTDMQSQMMALLLTANGHKVVTETVFENRFMHVAEFKRMNANINV
EGRSAKLEGKSQLQGAQVKATDLRAAAALILAGLVADGKTSVTELTHLDR
GYVDLHGKLKQLGADIERIND >HGS005, SEQ ID NO: 9, rho,
transcriptional terminator Rho TTCATGTATTTAAAAGGTTGGGGATTAGCA-
TAATGGGATTGTGCTAGCAC AGTTATTTATGCATTGTCATGCCTATCTATTACTTAC-
TAACTAAAAAATA ATGAAATGGGTGTAAACTATATGCCTGAAAGAGAACGTACATCT- CCTCAG
TATGAATCATTCCACGAATTGTACAAGAACTATACTACCAAGGAACTCAC
TCAAAAAGCTAAAACTCTTAAGTTGACGAACCATAGTAAATTAAATAAAA
AAGAACTTGTTCTAGCTATTATGGAAGCACAAATGGAAAAAGATGGTAAC
TATTATATGGAAGGTATCTTAGATGATATACAACCAGGTGGTTATGGTTT
TTTAAGAACAGTGAACTATTCTAAAGGGGAAAAAGATATTTATATATCTG
CTAGCCAAATTCGTCGTTTTGAAATTAAACGTGGGGATAAAGTAACTGGG
AAAGTTAGAAAACCTAAAGATAACGAAAAATATTATGGCTTATTACAAGT
TGACTTTGTCAATGACCATAACGCAGAAGAAGTGAAGAAACGTCCGCATT
TCCAAGCTTTGACACCACTTTATCCAGATGAGCGTATTAAATTAGAGACA
GAAATACAAAATTATTCAACGCGCATCATGGATTTAGTAACACCGATTGG
TTTAGGTCAACGTGGTTTAATAGTGGCGCCACCTAAAGCAGGTAAAACAT
CGTTATTAAAAGAAATAGCGAATGCAATCAGTACGAACAAACCAGATGCA
AAGCTATTTATTTTGTTAGTTGGCGAGCGTCCTGAAGAGGTAACAGATTT
AGAACGCTCAGTAGAAGCTGCTGAAGTCGTTCATTCAACGTTTGACGAAC
CACCAGAACACCATGTTAAAGTAGCTGAATTATTACTTGAACGTGCAAAG
CGTTTAGTAGAAATTGGGGAAGATGTCATTATTTTAATGGATTCTATAAC
GAGATTAGCACGCGCTTATAACTTAGTTATTCCACCAAGTGGTCGTACAT
TATCAGGTGGTTTAGATCCTGCATCTTTACACAAACCAAAAGCATTCTTC
GGTGCAGCGAGAAATATTGAAGCGGGTGGAAGTTTAACAATACTTGCAAC
TGCATTAGTTGATACGGGTTCACGTATGGACGATATGATTTACGAAGAAT
TTAAAGGAACAGGTAACATGGAGTTACATTTAGATCGTAAATTGTCTGAA
CGTCGTATCTTCCCTGCAATTGATATGGCAGAAGTTCAACGCGTAAAGAA
GAATTGTTGATAAGTAAATCTGAATTAGACACATTATGGCAATTAAGAAA
TCTATTCACTGACTCAACTGACTTTACTGAAAGATTTATTCGCAAACTTA
AAAGGTCTAAGAATAATGAAGATTTCTTCAAGCAGCTACAAAAGTCTGCA
GAAGAAAGTACTAAAACGGGTCGACCTATAATTTAATAAACATTATATAG
GGGCTTGCGTTTTGAATTAATTACCTTTATAATTACACAGTATTGGGTAA
AAACTCACAAATAACTCTGTTCCAGATGGTTCAGGG >HGS005, SEQ ID NO: 10,
Rho, transcriptional terminator Rho
MPERERTSPQYESFHELYKNYTTKELTQKAKTLKLTNHSKLNKKELVLAI
MEAQMEKDGNYYMEGILDDIQPGGYGFLRTVNYSKGEKDIYISASQIRRF
EIKRGDKVTGKVRKPKDNEKYYGLLQVDFVNDHNAEEVKKRPHFQALTPL
YPDERIKLETEIQNYSTRIMDLVTPIGLGQRGLIVAPPKAGKTSLLKEIA
NAISTNKPDAKLFILLVGERPEEVTDLERSVEAAEVVHSTFDEPPEHHVK
VAELLLERAKRLVEIGEDVIILMDSITRLARAYNLVIPPSGRTLSGGLDP
ASLHKPKAFFGAARNIEAGGSLTILATALVDTGSRMDDMIYEEFKGTGNM
ELHLDRKLSERRIFPAIDIGRSSTRKEELLISKSELDTLWQLRNLFTDST
DFTERFIRKLKRSKNNEDFFKQLQKSAEESTKTGRPII >HGS006, SEQ ID NO: 11,
rnpA, ribonuclease P protein component
GATCTTTTTTTTCGTTTAAATTAAGAATAAATAGAAATTTATGTTATAAG
CTCAATAGAAGTTTAAATATAGCTTCAATAAAAACGATAATAAGCGAGTG
ATGTTATTGGAAAAAGCTTACCGAATTAAAAAGAATGCAGATTTTCAGAG
AATATATAAAAAAGGTCATTCTGTAGCCAACAGACAATTTGTTGTATACA
CTTGTAATAATAAAGAAATAGACCATTTTCGCTTAGGTATTAGTGTTTCT
AAAAAACTAGGTAATGCAGTGTTAAGAAACAAGATTAAAAGAGCAATACG
TGAAAATTTCAAAGTACATAAGTCGCATATATTGGCCAAAGATATTATTG
TAATAGCAAGACAGCCAGCTAAAGATATGACGACTTTACAAATACAGAAT
AGTCTTGAGCACGTACTTAAAATTGCCAAAGTTTTTAATAAAAAGATTAA
GTAAGGATAGGGTAGGGGAAGGAAAACATTAACCACTCAACACATCCCGA
AGTCTTACCTCAGACAAACGTAAGACTGACCTTAGGGTTATAATAACTTA CTTT >HGS006,
SEQ ID NO: 12, RnpA, ribonuclease P protein component
MLLEKAYRIKKNADFQRIYKKGHSVANRQFVVYTCNNKEIDHFRLGIS- VS
KKLGNAVLRNKIKRAIRENFKVHKSHILAKDIIVIARQPAXDMTTLQIQN
SLEHVLKIAKVFNXKIK >HGS007M, SEQ ID NO: 13, dnaB, replicative DNA
helicase CAGCAAAAACTGGTGAAGGTGGTAAATT- GTTTGGGTCAGTAAGTACAAAA
CAAATTGCCGAAGCACTAAAAGCACAACATGATAT- TAAAATTGATAAACG
TAAAATGGATTTACCAAATGGAATTCATTCCCTAGGATATAC- GAATGTAC
CTGTTAAATTAGATAAGAAGTTGAAGGTACAATTCGCGTACACACAGTT- G
AACAATAAAGTTGGATTGAAATAAGAGGTGTAACCATTCATGGATAGAAT
GTATGAGCAAAATCAAATGCCGCATAACAATGAAGCTGAACAGTCTGTCT
TAGGTTCAATTATTATAGATCCAGAATTGATTAATACTACTCAGGAAGTT
TTGCTTCCTGAGTCGTTTTATAGGGGTGCCCATCAACATATTTTCCGTGC
AATGATGCACTTAAATGAAGATAATAAAGAAATTGATGTTGTAACATTGA
TGGATCAATTATCGACGGAAGGTACGTTGATgAAGCGGGTGGCCCGCAAT
ATCTTGCAGAGTTATCTACAAATGTACCAACGACGCGAAATGTTCAGTAT
TATACTGATATCGTTTCTAAGCATGCATTAAAACGTAGATTGATTCAAAC
TGCAGATAGTATTGCCAATGATGGATATAATGATGAACTTGAACTAGATG
CGATTTTAAGTGATGCAGAACGTCGAATTTTAGAGCTATCATCTTCTCGT
GAAAGCGATGGCTTTAAAGACATTCGAGACGTCTTAGGACAAGTGTATGA
AACAGCTGAAGAGCTTGATCAAAATAGTGGTCAAACACCAGGTATACCTA
CAGGATATCGAGATTTAGACCAAATGACAGCAGGGTTCAACCGAAATGAT
TTAATTATCCTTGCAGCGCGTCCATCTGTAGGTAAGACTGCGTTCGCACT
TAATATTGCACAAAAAGTTGCAACGCATGAAGATATGTATACAGTTGGTA
TTTTCTCGCTAGAGATGGGTGCTGATCAGTTAGCCACACGTATGATTTGT
AGTTCTGGAAATGTTGACTCAAACCGCTTAAGAACGGGTACTATGACTGA
GGAAGATTGGAGTCGTTTTACTATAGCGGTAGGTAAATTATCACGTACGA
AGATTTTTATTGATGATACACCGGGTATTCGAATTAATGATTTACGTTCT
AAATGTCGTCGATTAAAGCAAGAACATGGCTTAGACATGATTGTGATTGA
CTACTTACAGTTGATTCAAGGTAGTGGTTCACGTGCGTCCGATAACAGAC
AACAGGAAGTTTCTGAAATCTCTCGTACATTAAAAGCATTAGCCCGTGAA
TTAAAATGTCCAGTTATCGCATTAAGTCAGTTATCTCGTGGTGTTGAACA
ACGACAAGATAAACGTCCAATGATGAGTGATATTCGTGAATCTGGTTCGA
TTGAGCAAGATGCCGATATCGTTGCATTCTTATACCGTGATGATTACTAT
AACCGTGGCGGCGATGAAGATGATGACGATGATGGTGGTTTCGAGCCACA
AACGAATGATGAAAACGGTGAAATTGAAATTATCATTGCTAAGCAACGTA
ACGGTCCAACAGGCACAGTTAAGTTACATTTTATGAAACAATATAATAAA
TTTACCGATATCGATTATGCACATGCAGATATGATGTAAAAAAGTTTTTC
CGTACAATAATCATTAAGATGATAAAATTGTACGGTTTTTATTTTGTTCT GAACGGGTTG
>HGS007M, SEQ ID NO: 14, DnaB, replicative DNA helicase
MDRMYEQNQMPHNNEAEQSVLGSIIIDPELINTTQEVLLPE- SFYRGAHQH
IFRAMMHLNEDNKEIDVVTLMDQLSTEGTLNEAGGPQYLAELSTNVPT- TR
NVQYYTDIVSKHALKRRLIQTADSIANDGYNDELELDAILSDAERRILEL
SSSRESDGFKDIRDVLGQVYETAEELDQNSGQTPGIPTGYRDLDQMTAGF
NRNDLIILAARPSVGKTAFALNIAQKVATHEDMYTVGIFSLEMGADQLAT
RMICSSGNVDSNRLRTGTMTEEDWSRFTIAVGKLSRTKIFIDDTPGIRIN
DLRSKCRRLKQEHGLDMIVIDYLQLIQGSGSRASDNRQQEVSEISRTLKA
LARELKCPVIALSQLSRGVEQRQDKRPMMSDIRESGSIEQDADIVAFLYR
DDYYNRGGDEDDDDDGGFEPQTNDENGEIEIIIAKQRNGPTGTVKLHFMK QYNKFTDIDYAHADMM
>HGS008, SEQ ID NO: 15, fabD, malonyl CoA-acyl carrier protein
transacylase GTGGTTCCGTATTATTAGGATTGGAAGGTACTGTAGTTAAAGCACACGGT
AGTTCAAATGCTAAAGCTTTTTATTCTGCAATTAGACAAGCGAAAATCGC
AGGAGAACAAAATATTGTACAAACAATGAAAGAGACTGTAGGTGAATCAA
ATGAGTAAAACAGCAATTATTTTTCCGGGACAAGGTGCCCAAAAAGTTGG
TATGGCGCAAGATTTGTTTAACAACAATGATCAAGCAACTGAAATTTTAA
CTTCAGCAGCGAACACATTAGACTTTGATATTTTAGAGACAATGTTTACT
GATGAAGAAGGTAAATTGGGTGAAACTGAAAACACACAACCAGCTTTATT
GACGCATAGTTCGGCATTATTAGCAGCGCTAAAAAATTTGAATCCTGATT
TTACTATGGGGCATAGTTTAGGTGAATATTCAAGTTTAGTTGCAGCTGAC
GTATTATCATTTGAAGATGCAGTTAAAATTGTTAGAAAACGTGGTCAATT
AATGGCGCAAGCATTTCCTACTGGTGTAGGAAGCATGGCTGCAGTATTGG
GATTAGATTTTGATAAAGTCGATGAAATTTGTAAGTCATTATCATCTGAT
GACAAAATAATTGAACCAGCAAACATTAATTGCCCAGGTCAAATTGTTGT
TTCAGGTCACAAAGCTTTAATTGATGAGCTAGTAGAAAAAGGTAAATCAT
TAGGTGCAAAACGTGTCATGCCTTTAGCAGTATCTGGACCATTCCATTCA
TCGCTAATGAAAGTGATTGAAGAAGATTTTTCAAGTTACATTAATCAATT
TGAATGGCGTGATGCTAAGTTTCCTGTAGTTCAAAATGTAAATGCGCAAG
GTGAAACTGACAAAGAAGTAATTAAATCTAATATGGTCAAGCAATTATAT
TCACCAGTACAATTCATTAACTCAACAGAATGGCTAATAGACCAAGGTGT
TGATCATTTTATTGAAATTGGTCCTGGAAAAGTTTTATCTGGCTTAATTA
AAAAAATAAATAGAGATGTTAAGTTAACATCAATTCAAACTTTAGAAGAT
GTGAAAGGATGGAATGAAAATGACTAAGAGTGCTTTAGTAACAGGTGCAT
CAAGAGGAATTGGACGTAGTATTGCGTTACAATTAGCAGAAGAAGGATAT
AATGTAGCAGTAAACTATGC >HGS008, SEQ ID NO: 16, FabD, malonyl
CoA-acyl carrier protein transacylase
MSKTAIIFPGQGAQKVGMAQDLFNNNDQATEILTSAANTLDFDILETMFT
DEEGKLGETENTQPALLTHSSALLAALKNLNPDFTMGHSLGEYSSLVAAD
VLSFEDAVKIVRKRGQLMAQAFPTGVGSMAAVLGLDFDKVDEICKSLSSD
DKIIEPANINCPGQIVVSGHKALIDELVEKGKSLGAKRVMPLAVSGPFHS
SLMKVIEEDFSSYINQFEWRDAKFPVVQNVNAQGETDKEVIKSNMVKQLY
SPVQFINSTEWLIDQGVDHFIEIGPGKVLSGLIKKINRDVKLTSIQTLED VKGWNEND
>HGS009, SEQ ID NO: 17, aLf1, fructose-bisphosphate aldolase
AAATACACATTTAATCTGCAGTATTTCAATGCATT- GACGCTATTTTTTTG
ATATAATTACTTTGAAAAATACGTGCGTAAGCACTCAAGGAG- GAACTTTC
ATGCCTTTAGTTTCAATGAAAGAAATGTTAATTGATGCAAAAGAAAATG- G
TTATGCGGTAGGTCAATACAATATTAATAACCTAGAATTCACTCAAGCAA
TTTTAGAAGCGTCACAAGAAGAAAATGCACCTGTAATTTTAGGTGTTTCT
GAAGGTGCTGCTCGTTACATGAGCGGTTTCTACACAATTGTTAAAATGGT
TGAAGGGTTAATGCATGACTTAAACATCACTATTCCTGTAGCAATCCATT
TAGACCATGGTTCAAGCTTTGAAAAATGTAAAGAAGCTATCGATGCTGGT
TTCACATCAGTAATGATCGATGCTTCACACAGCCCATTCGAAGAAAACGT
AGCAACAACTAAAAAAGTTGTTGAATACGCTCATGAAAAAGGTGTTTCTG
TAGAAGCTGAATTAGGTACTGTTGGTGGACAAGAAGATGATGTTGTAGCA
GACGGCATCATTTATGCTGATCCTAAAGAATGTCAAGAACTAGTTGAAAA
AACTGGTATTGATGCATTAGCGCCAGCATTAGGTTCAGTTCATGGTCCAT
ACAAAGGTGAACCAAAATTAGGATTPAAAGAAATGGAAGAAATCGGTTTA
TCTACAGGTTTACCATTAGTATTACACGGTGGTACTGGTATCCCGACTAA
AGATATCCAAAAAGCAATTCCATTTGGTACAGCTAAAATTAACGTAAACA
CTGAAAACCAAATCGCTTCAGCAAAAGCAGTTCGTGACGTTTTAAATAAC
GACAAAGAAGTTTACGATCCTCGTAAATACTTAGGACCTGCACGTGAAGC
CATCAAAGAAACAGTTAAAGGTAAAATTAAAGAGTTCGGTACTTCTAACC
GCGCTAAATAATTAATATTTAGTCTTTAAGTTATTAATAACGTAGGGATA
TTAATTTTAAAAGAAGCAGACAAAATGGTGTTTGCTTCTTTTTTATGTCG
TATAAGTAATAAATAAAACAGTTTGATTTT >HGS009, SEQ ID NO: 18, Alf1,
fructose-bisphosphate aldolase
MPLVSMKEMLIDAKENGYAVGQYNINNLEFTQAILEASQEENAPVILGVS
EGAARYMSGFYTIVKMVEGLMHDLNITIPVAIHLDHGSSFEKCKEAIDAG
FTSVMIDASHSPFEENVATTKKVVEYAHEKGVSVEAELGTVGGQEDDVVA
DGIIYADPKECQELVEKTGIDALAPALGSVHGPYKGEPKLGFKEMEEIGL
STGLPLVLHGGTGIPTKDIQKAIPFGTAXINVNTENQIASAKAVRDVLNN
DKEVYDPRKYLGPAREAIKETVKGKIKEFGTSNRAK >HGS014, SEQ ID NO: 19
GCTATAATAGGCATGGTTACAATGAGCTTGCTCATACATATTAATATAAT
TACAAAAACACGTCGGAGGTACGACATGATTAAAAATACAATTAAAAAAT
TGATAGAACATAGTATATATACGACTTTTAAATTACTATCAAAATTGCCA
AACAAGAATCTAATTTATTTTGAAAGCTTTCATGGTAAACAATACAGCGA
CAACCCCAAAGCATTATATGAATACTTAACTGAACATAGCGATGCCCAAT
TAATATGGGGTGTGAAAAAAGGATATGAACACATATTCCAACAGCACAAT
GTACCATATGTTACAAAGTTTTCAATGAAATGGTTTTTAGCGATGCCAAG
AGCGAAAGCGTGGATGATTAACACACGTACACCAGATTGGTTATATAAAT
CACCGCGAACGACGTACTTACAAACATGGCATGGCACGCCATTAAAAAAG
ATTGGTTTGGATATTAGTAACGTTAAAATGCTAGGAACAAATACTCAAAA
TTACCAAGATGGCTTTAAAAAAGAAAGCCAACGGTGGGATTATCTAGTGT
CACCTAATCCATATTCGACATCGATATTTCAAAATGCATTTCATGTTAGT
CGAGATAAGATTTTGGAAACAGGTTATCCAAGAAATGATAAATTATCACA
TAAACGCAATGATACTGAATATATTAATGGTATTAAGACAAGATTAAATA
TTCCATTAGATAAAAAAGTGATTATGTACGCGCCAACTTGGCGTGACGAT
GAAGCGATTCGAGAAGGTTCATATCAATTTAATGTTAACTTTGATATAGA
AGCTTTGCGTCAAGCGCTGGATGATGATTATGTTATTTTATTACGCATGC
ATTATTTAGTTGTGACACGTATTGATGAACATGATGATTTTGTGAAAGAC
GTTTCAGATTATGAAGACATTTCGGATTTATACTTAATCAGCGATGCGTT
AGTTACCGACTACTCATCTGTCATGTTCGACTTCGGTGTATTAAAGCGTC
CGCAAATTTTCTATGCATATGACTTAGATAAATATGGCGATGAGCTTAGA
GGTTTTTACATGGATTATAAAAAAGAGTTGCCAGGTCCAATTGTTGAAAA
TCAAACAGCACTCATTGATGCATTAAAACAAATCGATGAGACTGCAAATG
AGTATATTGAAGCACGAACGGTATTTTATCAAAAATTCTGTTCATTAGAA
GATGGACAAGCGTCACAACGAATTTGCCAAACGATTTTTAAGTGATAACT
TAAAAACAATAAAAAATTATAAATTAATTAGTTAAGTGATATAAATAATA
ACGAAATGTTTGCTTGTATGTTATTATTTGTGTATGAAA >HGS014, SEQ ID NO: 20
MIKNTIKKLIEHSIYTTFKLLSKLPNKNLIYFESFHGKQYSDNPKALYEY
LTEHSDAQLIWGVKXGYEHIFQQHNVPYVTKFSMKWFLAMPRAKAWMINT
RTPDWLYKSPRTTYLQTWHGTPLKKIGLDISNVKMLGTNTQNYQDGFKKE
SQRWDYLVSPNPYSTSIFQNAFHVSRDKILETGYPRNDKLSHKRNDTEYI
NGIKTRLNIPLDKKVIMYAPTWRDDEAIREGSYQFNVNFDIEALRQALDD
DYVILLRMHYLVVTRIDEHDDFVKDVSDYEDISDLYLISDALVTDYSSVM
FDFGVLKRPQIFYAYDLDKYGDELRGFYMDYKKELPGPIVENQTALIDAL
KQIDETANEYIEARTVFYQKFCSLEDGQA5QRICQTIFK >HGS016, SEQ ID NO: 21,
murA, UDP-N-acetylglucosamine 1-carboxyvinyltransferase
TGATTTGTAATCAAAACTAGATATAATTAAATAATGACTTAAAATAATTT
TAAAATAGGGAAATGTAAAGTAATAGGAGTTCTAAGTGGAGGATTTACGA
TGGATAAAATAGTAATCAAAGGTGGAAATAAATTAACGGGTGAAGTTAAA
GTAGAAGGTGCTAAAAATGCAGTATTACCAATATTGACAGCATCTTTATT
AGCTTCTGATAAACCGAGCAAATTAGTTAATGTTCCAGCTTTAAGTGATG
TAGAAACAATAAATAATGTATTAACAACTTTAAATGCTGACGTTACATAC
AAAAAGGACGAAAATGCTGTTGTCGTTGATGCAACAAAGACTCTAAATGA
AGAGGCACCATATGAATATGTTAGTAAAATGCGTGCAAGTATTTTAGTTA
TGGGACCTCTTTTAGCAAGACTAGGACATGCTATTGTTGCATTGCCTGGT
GGTTGTGCAATTGGAAGTAGACCGATTGAGCAACACATTAAAGGTTTTGA
AGCTTTAGGCGCAGAAATTCATCTTGAAAATGGTAATATTTATGCTAATG
CTAAAGATGGATTAAAAGGTACATCAATTCATTTAGATTTTCCAAGTGTA
GGAGCAACACAAAATATTATTATGGCAGCATCATTAGCTAAGGGTAAGAC
TTTAATTGAAAATGCAGCTAAAGAACCTGAAATTGTCGATTTAGCAAACT
ACATTAATGAAATGGGTGGTAGAATTACTGGTGCTGGTACAGACACAATT
ACAATCAATGGTGTAGAATCATTACATGGTGTAGAACATGCTATCATTCC
AGATAGAATTGAAGCAGGCACATTACTAATCGCTGGTGCTATAACGCGTG
GTGATATTTTTGTACGTGGTGCAATCAAAGAACATATGGCGAGTTTAGTC
TATAAACTAGAAGAAATGGGCGTTGAATTGGACTATCAAGAAGATGGTAT
TCGTGTACGTGCTGAAGGGGAATTACAACCTGTAGACATCAAAACTCTAC
CACATCCTGGATTCCCGACTGATATGCAATCACAAATGATGGCATTGTTA
TTAACGGCAAATGGTCATAAAGTCGTAACCGAAACTGTTTTTGAAAACCG
TTTTATGCATGTTGCAGAGTTCAAACGTATGAATGCTAATATCAATGTAG
AAGGTCGTAGTGCTAAACTTGAAGGTAAAAGTCAATTGCAAGGTGCACAA
GTTAAAGCGACTGATTTAAGAGCAGCAGCCGCCTTAATTTTAGCTGGATT
AGTTGCTGATGGTAAAACAAGCGTTACTGAATTAACGCACCTAGATAGAG
GCTATGTTGACTTACACGGTAAATTGAAGCAATTAGGTGCAGACATTGAA
CGTATTAACGATTAATTCAGTAAATTAATATAATGGAGGATTTCAACCAT
GGAAACAATTTTTGATTATAACCAAATTAA >HGS016, SEQ ID NO: 22, MurA,
UDP-N-acetylglucosamine 1-carboxyvinyltransferase
MDKIVIKGGNKLTGEVKVEGAKNAVLPILTASLLASDKPSKLVNVPALSD
VETINNVLTTLNADVTYKKDENAVVVDATKTLNEEAPYEYVSKMRASILV
MGPLLARLGHAIVALPGGCAIGSRPIEQHIKGFEALGAEIHLENGNIYAN
AKDGLKGTSIHLDFPSVGATQNIIMAASLAXGKTLIENAAKEPEIVDLAN
YINEMGGRITGAGTDTITINGVESLHGVEHAIIPDRIEAGTLLIAGAITR
GDIFVRGAIKEHMASLVYKLEEMGVELDYQEDGIRVRAEGELQPVDIKTL
PHPGFPTDMQSQMMALLLTANGHKVVTETVFENRFMHVAEFKRMNANINV
EGRSAKLEGKSQLQGAQVKATDLRAAAALILAGLVADGKTSVTELTHLDR
GYVDLHGKLKQLGADIERIND >HGS018, SEQ ID NO: 23, dnaJ, DNA ligase
AGAAAAATGGCTCAATCGAACTAGATATTATCTTTAAATCACAAGGGCCA
AAACGTTTGTTAGCGCAATTTGCACCAATTGAAAAAAGGAGGATTAAGGG
ATGGCTGATTTATCGTCTCGTGTGAACGAGTTACATGATTTATTAAATCA
ATACAGTTATGAATACTATGTAGAGGATAATCCATCTGTACCAGATAGTG
AATATGACAAATTACTTCATGAACTGATTAAAATAGAAGAGGAGCATCCT
GAGTATAAGACTGTAGATTCTCCAACAGTTAGAGTTGGCGGTGAAGCCCA
AGCCTCTTTCAATAAAGTCAACCATGACACGCCAATGTTAAGTTTAGGGA
ATGCATTTAATGAGGATGATTTGAGAAAATTCGACCAACGCATACGTGAA
CAAATTGGCAACGTTGAATATATGTGCGAATTAAAAATTGATGGCTTAGC
AGTATCATTGAAATATGTTGATGGATACTTCGTTCAAGGTTTAACACGTG
GTGATGGAACAACAGGTGAAGATATTACCGAAAATTTAAAAACAATTCAT
GCGATACCTTTGAAAATGAAAGAACCATTAAATGTAGAAGTTCGTGGTGA
AGCATATATGCCGAGACGTTCATTTTTACGATTAAATGAAGAAAAAGAAA
AAAATGATGAGCAGTTATTTGCAAATCCAAGAAACGCTGCTGCGGGATCA
TTAAGACAGTTAGATTCTAAATTAACGGCAAAACGAAAGCTAAGCGTATT
TATATATAGTGTCAATGATTTCACTGATTTCAATGCGCGTTCGCAAAGTG
AAGCATTAGATGAGTTAGATAAATTAGGTTTTACAACGAATAAAAATAGA
GCGCGTGTAAATAATATCGATGGTGTTTTAGAGTATATTGAAAAATGGAC
AAGCCAAAGAGAGTCATTACCTTATGATATTGATGGGATTGTTATTAAGG
TTAATGATTTAGATCAACAGGATGAGATGGGATTCACACAAAAATCTCCT
AGATGGGCCATTGCTTATAAATTTCCAGCTGAGGAAGTAGTAACTAAATT
ATTAGATATTGAATTAAGTATTGGACGAACAGGTGTAGTCACACCTACTG
CTATTTTAGAACCAGTAAAAGTRGCTGGTACAACTGTATCAAGAGCATCT
TTGCACAATGAGGATTTAATTCATGACAGAGATATTCGAATTGGTGATAG
TGTTGTAGTGAAAAAAGCAGGTGACATCATACCTGAAGTTGTACGTAGTA
TTCCAGAACGTAGACCTGAGGATGCTGTCACATATCATATGCCAACCCAT
TGTCCAAGTTGTGGACATGAATTAGTACGTATTGAAGGCGAAGTAGCACT
TCGTTGCATTAATCCAAAATGCCAAGCACAACTTGTTGAAGGATTGATTC
ACTTTGTATCAAGACAAGCCATGAATATTGATGGTTTAGGCACTAAAATT
ATTCAACAGCTTTATCAAAGCGAATTAATTAAAGATGTTGCTGATATTTT
CTATTTAACAGAAGAAGATTTATTACCTTTAGACAGAATGGGGCAGAAAA
AAGTTGATAATTTATTAGCTGCCATTCAACAAGCTAAGGACAACTCTTTA
GAAAATTTATTATTTGGTCTAGGTATTAGGCATTTAGGTGTTAAAGCGAG
CCAAGTGTTAGCAGAAAAATATGAAAGCATAGATCGATTACTAACGGTAA
CTGAAGCGGAATTAGTAGAAATTCATGATATAGGTGATAAAGTAGCACAA
TCTGTAGTTACTTATTTAGAAAATGAAGATATTCGTGCTTTAATTCAAAA
ATTAAAAGATAAACATGTTAATATGATTTATAAAGGTATCAAAACATCAG
ATATTGAAGGACATCCTGAATTTAGTGGTAAAACGATAGTACTGACTGGT
AAGYTACATCAAATGACACGCAATGAAGCATCTAAATGGCTTGCATCACA
AGGTGCTAAAGTTACAAGTAGCGTTACTAAAAATACAGATGTCGTTATTG
CTGGTGAAGATGCAGGTTCAAAATTAACAAAAGCACAAAGTTTAGGTATT
GAAATTTGGACAGAGCAACAATTTGTAGATAAGCAAAATGAATTAAATAG
TTAGAGGGGTATGTCGATGAAGCGTACATTAGTATTATTGATTACAGCTA
TCTTTATACTCGCTGCTTGTGGTAACCATAAGGATGACCAGGCTGGAAAA GATA >HGS018,
SEQ ID NO: 24, DnaJ, DNA ligase
MADLSSRVNELHDLLNQYSYEYYVEDNPSVPDSEYDKLLHELIKIEEEHP
EYKTVDSPTVRVGGEAQASFNKVNHDTPMLSLGNAFNEDDLRKFDQRIRE
QIGNVEYMCELKIDGLAVSLKYVDGYFVQGLTRGDGTTGEDITENLKTIH
AIPLKMKEPLNVEVRGEAYMPRRSFLRLNEEKEKNDEQLFANPRNAAAGS
LRQLDSKLTAKRXLSVFIYSVNDFTDFNARSQSEALDELDKLGFTTNXNR
ARVNNIDGVLEYIEKWTSQRESLPYDIDGIVIKVNDLDQQDEMGFTQKSP
RWAIAYKFPAEEVVTKLLDIELSIGRTGVVTPTAILEPVKVAGTTVSRAS
LHNEDLIHDRDIRIGDSVVVKKAGDIIPEVVRSIPERRPEDAVTYHMPTH
CPSCGHELVRIEGEVALRCINPKCQAQLVEGLIHFVSRQAMNIDGLGTKI
IQQLYQSELIKDVADIFYLTEEDLLPLDRMGQKKVDNLLAAIQQAKDNSL
ENLLFGLGIRHLGVKASQVLAEKYETIDRLLTVTEAELVEIHDIGDKVAQ
SVVTYLENEDIRALIQKLKDKHVNMIYKGIKTSDIEGHPEFSGKTIVLTG
KLHQMTRNEASKWLASQGAKVTSSVTKNTDVVIAGEDAGSKLTKAQSLGI
EIWTEQQFVDKQNELNS >HGS019, SEQ ID NO: 25, mapM, methionine
aminopeptidase TGTCTCACTCACTTTCCAAAATACTAAAGTAACATCTTTA- GTATATCAAA
GAATTTTTGCTATAATAAGTTATAATTATATAAAAAAGGAACGGGAT- AAA
ATGATTGTAAAAACAGAAGAAGAATTACAAGCGTTAAAAGAAATTGGATA
CATATGCGCTAAAGTGCGCAATACAATGCAAGCTGCAACCAAACCAGGTA
TCACTACGAAAGAGCTTGATAATATTGCGAAAGAGTTATTTGAAGAATAC
GGTGCTATTTCTGCGCCAATTCATGATGAAAATTTTCCTGGTCAAACGTG
TATTAGTGTCAATGAAGAGGTGGCACATGGGATTCCAAGTAAGCGTGTCA
TTCGTGAAGGAGATTTAGTAAATATTGATGTATCGGCTTTGAAGAATGGC
TATTATGCAGATACAGGCATTTCATTTGTCGTTGGAGAATCAGATGATCC
AATGAAACAAAAAGTATGTGACGTAGCAACGATGGCATTTGAGAATGCAA
TTGCAAAAGTAAAACCGGGTACTAAGTTAAGTAACATTGGTAAAGCGGTG
CATAATACAGCTAGACAAAATGATTTGAAAGTCATTAAAAACTTAACAGG
TCATGGTGTTGGTTTATCATTACATGAAGCACCAGCACATGTACTTAATT
ACTTTGATCCAAAAGACAAAACATTATTAACTGAAGGTATGGTATTAGCT
ATTGAACCGTTTATCTCATCAAATGCATCATTTGTTACAGAAGGTAAAAA
TGAATGGGCTTTTGAAACGAGCGATAAAAGTTTTGTTGCTCAAATTGAGC
ATACGGTTATCGTGACTAAGGATGGTCCGATTTTAACGACAAAGATTGAA
GAAGAATAGTTCAACATATACTAAGACTAAAGTATGAACATCATTTAGTT
CCGGAGCCTATTGGTTTCGGAACTGTTTTATAATAATTAAGAACACAATC AAT >HGS019,
SEQ ID NO: 26, MapM, methionine aminopeptidase
MIVKTEEELQALKEIGYICAKVRNTMQAATKPGITTKELDNIAKELFEEY
GAISAPIHDENFPGQTCISVNEEVAHGIPSKRVIREGDLVNIDVSALKNG
YYADTGISFVVGESDDPMKQKVCDVATMAFENAIAKVKPGTKLSNIGKAV
HNTARQNDLKVIKNLTGHGVGLSLHEAPAHVLNYFDPKDKTLLTEGMVLA
IEPFISSNASFVTEGKNEWAFETSDKSFVAQIEHTVIVTKDGPILTTKIE EE
>HGS022-23-24, SEQ ID NO: 27, adt, glutamyl-tRNA
amidotransferase subunit a, b, and c (operon comprising three ORFs
listed below) TATACAGTTTATATGAAATTAAAGTAGCACCTCATAAATACTTAGATTTT
TAATTGGAAATTTGATACAATTTAGTGATGAATGACTTAAAGGAGGCTTT
TATTAATGACAAAAGTAACACGTGAAGAAGTTGAGCATATCGCGAATCTT
GCAAGACTTCAAATTTCTCCTGAAGAAACGGAAGAAATGGCCAACACATT
AGAAAGCATTTTAGATTTTGCAAAACAAAATGATAGCGCTGATACAGAAG
GCGTTGAACCTACATATCACGTTTTAGATTTACAAAAACGTTTTACGTGA
AGATAAGCAATTAAAGGTATTCCACAAGAATTAGCTTTGAAAAATGCCAA
AGAAACAGAAGATGGACAATTTAAAGTGCCTACAATCATGAATGAGGAGG
ACGCGTAAGATGAGCATTCGCTACGAATCGGTTGAGAATTTATTAACTTT
AATAAAAGACAAAAAAATCAAACCATCTGATGTTGTTAAAGATATATATG
ATGCAATTGAAGAGACTGATCCAACAATTAAGTCTTTTCTAGCGCTGGAT
AAAGAAAATGCAATCAAAAAAGCGCAAGAATTGGATGAATTACAAGCAAA
AGATCAAATGGATGGCAAATTATTTGGTATTCCAATGGGTATAAAAGATA
ACATTATTACAAACGGATTAGAAACAACATGTGCAAGTAAAATGTTAGAA
GGTTTTGTGCCAATTTACGAATCTACTGTAATGGAAAAACTACATAATGA
AAATGCCGTTTTAATCGGTAAATTAAATATGGATGAGTTTGCAATGGGTG
GTTCAACAGAAACATCTTATTTCAAAAAAACAGTTAACCCATTTGACCAT
AAAGCAGTGCCAGGTGGTTCATCAGGTGGATCTGCAGCAGCAGTTGCAGC
TGGCTTAGTACCATTTAGCTTAGGTTCAGACACAGGTGGTTCAATTAGAC
AACCGGCTGCATATTGTGGCGTTGTCGGTATGAAACCAACATACGGTCGT
GTATCTCGATTTGGATTAGTTGCTTTTGCATCTTCATTAGACCAAATTGG
TCCATTGACTCGAAATGTAAAAGATAATGCAATCGTATTAGAAGCTATTT
CTGGTGCAGATGTTAATGACTCTACAAGTGCACCAGTTGATGATGTAGAC
TTTACATCTGAAATTGGTAAAGATATTAAAGGATTAAAAGTTGCATTACC
TAAAGAATACTTAGGTGAAGGTGTAGCTGATGACGTAAAAGAAGCAGTTC
AAAACGCTGTAGAAACTTTAAAATCTTTAGGTGCTGTCGTTGAGGAAGTA
TCATTGCCAAATACTAAATTTGGTATTCCATCATATTACGTGATTGCATC
ATCAGAAGCTTCGTCAAACCTTTCTCGTTTTGACGGAATTCGTTATGGTT
ATCATTCTAAAGAAGCTCATTCATTAGAAGAATTATATAAAATGTCAAGA
TCTGAAGGTTTCGGTAAAGAAGTAAAACGTCGTATTTTCTTAGGTACATT
TGCATTAAGTTCAGGTTACTATGATGCTTACTATAAAAAATCTCAAAAAG
TTAGAACATTGATTAAAAATGACTTTGATAAAGTATTCGAAAATTATGAT
GTAGTAGTTGGTCCAACAGCGCCTACAACTGCGTTTAATTTAGGTGAAGA
AATTGATGATCCATTAACAATGTATGCCAATGATTTATTAACAACACCAG
TAAACTTAGCTGGATTACCTGGTATTTCTGTTCCTTGTGGACAATCAAAT
GGCCGACCAATCGGTTTACAGTTCATTGGTAAACCATTCGATGAAAAAAC
GTTATATCGTGTCGCTTATCAATATGAAACACAATACAATTTACATGACG
TTTATGAAAAATTATAAGGAGTGGAAATCATGCATTTTGAAACAGTTATA
GGACTTGAAGTTCACGTAGAGTTAAAAACGGACTCAAAAATGTTTTCTCC
ATCACCAGCGCATTTTGGAGCAGAACCTAACTCAAATACAAATGTTATCG
ACTTAGCATATCCAGGTGTCTTACCAGTTGTTAATAAGCGTGCAGTAGAC
TGGGCAATGCGTGCTGCAATGGCACTAAATATGGAAATCGCAACAGAATC
TAAGTTTGACCGTAAGAACTATTTCTATCCAGATAATCCAAAAGCATATC
AAATTTCTCAATTTGATCAACCAATTGGTGAAAATGGATATATCGATATC
GAAGTCGACGGTGAAACAAAACGAATCGGTATTACTCGTCTTCACATGGA
AGAAGATGCTGGTAAGTCAACACATAAAGGTGAGTATTCATTAGTTGACT
TGAACCGTCAAGGTACACCGCTAATTGAAATCGTATCTGAACCAGATATT
CGTTCACCTAAAGAAGCATATGCATATTTAGAAAAATTGCGTTCAATTAT
TCAATACACTGGTGTATCAGACGTTAAGATGGAAGAGGGATCTTTACGTT
GTGATGCTAACATCTCTTTACGTCCATATGGTCAAGAAAAATTTGGTACT
AAAGCCGAATTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTT
AGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAA
TCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTA
ATGCGTGTTAAAGAAGGTTCTGATGATTACCGTTACTTCCCAGAGCCTGA
CATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGA
CAATTCCTGAATTACCAGATGAACGTAAAGCTAAGTATGTAAATGAATTA
GGTTTACCTGCATACGATGCACACGTATTAACATTGACTAAAGAAATGTC
AGATTTCTTTGAATCAACAATTGAACACGGTGCAGATGTTAAATTAACAT
CTAACTGGTTAATGGGTGGCGTAAACGAATATTTAAATAAAAATCAAGTA
GAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAA
ACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCC
CAGAGTTAGCAGCTAAAGGTGGTAATGCTAAACAGATTATGGAAGATAAT
GGCTTAGTTCAAATTTCTGATGAAGCAACACTTCTAAAATTTGTAAATGA
AGCATTAGACAATAACGAACAATCAGTTGAAGATTACAAAAATGGTAAAG
GCAAAGCTATGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGT
CAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAA
AAGATAATTTAAATCATCAAACTATGAAGATTTAAAAAATAAACCCTTGA
TTGCTGACTTAGATGCAATCGAGGGTTTATTTATATCTATAGAAGTCAAA >HGS022, SEQ
ID NO: 28, Adt, glutamyl-tRNA amidotransferase subunit a
MSIRYESVENLLTLIKDKXIKPSDVVKDIYDAIEETDPTIKSFLALDREN
AIKKAQELDELQAKDQMDGKLFGIPMGIKDNIITNGLETTCASKMLEGFV
PIYESTVMEKLHNENAVLIGKLNMDEFAMGGSTETSYFKKTVNPFDHKAV
PGGSSGGSAAAVAAGLVPFSLGSDTGGSIRQPAAYCGVVGNKPTYGRVSR
FGLVAFASSLDQIGPLTRNVKDNAIVLEAISGADVNDSTSAPVDDVDFTS
EIGKDIKGLKVALPKEYLGEGVADDVKEAVQNAVETLKSLGAVVEEVSLP
NTKFGIPSYYVIASSEASSNLSRFDGIRYGYHSKEAHSLEELYKMSRSEG
FGKEVKRRIFLGTFALSSGYYDAYYKKSQKVRTLIKNDFDKVFENYDVVV
GPTAPTTAFNLGEEIDDPLTMYANDLLTTPVNLAGLPGISVPCGQSNGRP
IGLQFIGKPFDEKTLYRVAYQYETQYNLHDVYEKL >HGS023, SEQ ID NO: 29, Adt,
glutamyl-tRNA amidotransferase subunit b
MHFETVIGLEVHVELKTDSKMFSPSPAHFGAEPNSNTNVIDLAYPGVLPV
VNKRAVDWAMRAAMALNMEIATESKFDRKNYFYPDNPKAYQISQFDQPIG
ENGYIDIEVDGETKRIGITRLHMEEDAGKSTHKGEYSLVDLNRQGTPLIE
IVSEPDIRSPKEAYAYLEKLRSIIQYTGVSDVKMEEGSLRCDANISLRPY
GQEKFGTKAELKNLNSFNYVRKGLEYEEKRQEEELLUGGEIGQETRRFDE
STGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERK
AKYVNELGLPAYDAHVLTLTKEMSDFFESTIEHGADVKLTSNWLMGGVNE
YLNKNQVELLDTKLTPENLAGMIKLIEDGTMSSKIAKKVFPELAAKGGNA
KQIMEDNGLVQISDEATLLKFVNEALDNNEQSVEDYKNGKGKAMGFLVGQ
IMKASKGQANPQLVNQLLKQELDKR >HGS024, SEQ ID NO: 30, Adt,
glutamyl-tRNA amidotransferase subunit c
MTKVTREEVEHIANLARLQISPEETEEMANTLESILDFAKQNDSADTEGV
EPTYHVLDLQNVLREDKAIKGIPQELALKNAKETEDGQFKVPTIMNEEDA >HGS025, SEQ
ID NO: 31, pth, peptidyl-tRNA hydrolase
CTTACTAAGCTAAAGAATAATGATAATTGATGGCAATGGCGGAAAATGGA
TGTTGTCATTATAATAATAAATGAAACAATTATGTTGGAGGTAAACACGC
ATGAAATGTATTGTAGGTCTAGGTAATATAGGTAAACGTTTTGAACTTAC
AAGACATAATATCGGCTTTGAAGTCGTTGATTATATTTTAGAGAAAAATA
ATTTTTCATTAGATAAACAAAAGTTTAAAGGTGCATATACAATTGAACGA
ATGAACGGCGATAAAGTGTTATTTATCGAACCAATGACAATGATGAATTT
GTCAGGTGAAGCAGTTGCACCGATTATGGATTATTACAATGTTAATCCAG
AAGATTTAATTGTCTTATATGATGATTTAGATTTAGAACAAGGACAAGTT
CGCTTAAGACAAAAAGGAAGTGCGGGCGGTCACAATGGTATGAAATCAAT
TATTAAAATGCTTGGTACAGACCAATTTAAACGTATTCGTATTGGTGTGG
GAAGACCAACGAATGGTATGACGGTACCTGATTATGTTTTACAACGCTTT
TCAAATGATGAAATGGTAACGATGGAAAAAGTTATCGAACACGCAGCACG
CGCAATTGAAAAGTTTGTTGAAACATCACGATTTGACCATGTTATGAATG
AATTTAATGGTGAAGTGAAATAATGACAATATTGACAACGCTTATAAAAG
AAGATAATCATTTTCAAGACCTTAATCAGGTATTTGGACAAGCAAACACA
CTAGTAACTGGTCTTTCCCCGT >HGS025, SEQ ID NO: 32, Pth,
peptidyl-tRNA hydrolase MKCCIVGLGNIGKRFELTR1NIGFEVVDYILEKNNFS-
LDKQKFKGAYTIE RNGDKVLFIEPMTMMNLSGEAVAPIMDYYNVNPEDLIVLYDDLD- LEQGQV
RLRQKGSAGGHNGMXSIIKMLGTDQFKRIRIGVGRPTNGMTVPDYVLQRF
SNDEMVTMEKVIEHAARAIEKFVETSRFDHVMNEFNGEVK >HGS026, SEQ ID NO: 33
TGATCCGATTATCTTAGTAGGTGCCAATGAAAGTTATGAG- CCACGTTGTC
GCGCGCACCATATCGTAGCACCTAGTGATAATAATAAGGAGGAATTA- TAA
GTGTTTGATCAATTAGATATTGTAGAAGAAAGATACGAACAGTTAAATGA
ACTGTTAAGTGACCCAGATGTTGTAAATGATTCAGATAAATTACGTAAAT
ATTCTAAAGAGCAAGCTGATTTACAAAAAACTGTAGATGTTTATCGTAAC
TATAAAGCTAAAAAAGAAGAATTAGCTGATATTGAAGAAATGTTAAGTGA
GACTGATGATAAAGAAGAAGTAGAAATGTTAAAAGAGGAGAGTAATGGTA
TTAAAGCTGAACTTCCAAATCTTGAAGAAGAGCTTAAAATATTATTGATT
CCTAAAGATCCTAATGATGACAAAGACGTTATTGTAGAAATAAGAGCAGC
AGCAGGTGGTGATGAGGCTGCGATTTTTGCTGGTGATTTAATGCGTATGT
ATTCAAAGTATGCTGAATCACAAGGATTCAAAACTGAAATAGTAGAAGCG
TCTGAAAGTGACCATGGTGGTTACAAAGAAATTAGTTTCTCAGTTTCTGG
TAATGGCGCGTATAGTAAATTGAAATTTGAAAATGGTGCGCACCGCGTTC
AACGTGTGCCTGAAACAGAATCAGGTGGACGTATTCATACTTCAACAGCT
ACAGTGGCAGTTTTACCAGAAGTTGAAGATGTAGAAATTGAAATTAGAAA
TGAAGATTTAAAAATCGACACGTATCGTTCAAGTGGTGCAGGTGGTCAGC
ACGTAAACACAACTGACTCTGCAGTACGTATTACCCATTTACCAACTGGT
GTCATTGCAACATCTTCTGAGAAGTCTCAAATTCAAAACCGTGAAAAAGC
AATGAAAGTGTTAAAAGCACGTTTATACGATATGAAAGTTCAAGAAGAAC
AACAAAAGTATGCGTCACAACGTAAATCAGCAGTCGGTACTGGTGATCGT
TCAGAACGTATTCGAACTTATAATTATCCACAAAGCCGTGTAACAGACCA
TCGTATAGGTCTAACGCTTCAAAAATTAGGGCAAATTATGGAAGGCCATT
TAGAAGAAATTATAGATGCACTGACTTTATCAGAGCAGACAGATAAATTG
AAAGAACTTAATAATGGTGAATTATAAAGAAAAGTTAGATGAAGCAATTC
ATTTAACACAACAAAAAGGGTTTGAACAAACACGAGCTGAATGGTTAATG
TTAGATGTATTTCAATGGACGCGTACG >HGS026, SEQ ID NO: 34
VFDQLDIVEERYEQLNBLLSDPDVVNDSDKLRKYSKEQADLQKTVDVYRN
YKAKKEELADIEEMLSETDDKEEVEMLKEESNGIKAELPNLEEELRILLI
PKDPNDDKDVIVEIRAAAGGDEAAIFAGDLMRMYSKYAESQGFKTEIVEA
SESDHGGYKEISFSVSGNGAYSKLKFENGAHRVQRVPETESGGRIHTSTA
TVAVLPEVEDVEIEIRNEDLKIDTYRSSGAGGQHVNTTDSAVRITHLPTG
VIATSSEKSQIQNREKAMKVLKARLYDNKVQEEQQKYASQRKSAVGTGDR
SERIRTYNYPQSRVTDHRIGLTLQKLGQIMEGHLEEIIDALTLSEQTDKL KELNNGEL
>HGS028, SEQ ID NO: 35
ATTTCTTAACATTGTTATTTAACAAAATTATGTTAAAATTTAGCATTATA
AAAGATGCAAATCAATGACTTGAATTGAAATATAAATAGGAGCGAATGCT
ATGGAATTATCAGAAATCAAACGAAATATAGATAAGTATAATCAAGATTT
AACACAAATTAGGGGGTCTCTTGACTTAGAGAACAAAGAAACTAATATTC
AAGAATATGAAGAAATGATGGCAGAACCTAATTTTTGGGATAACCAAACG
AAAGCGCAAGATATTATAGATAAAAATAATGCGTTAAAAGCAATAGTTAA
TGGTTATAAAACACTACAAGCAGAAGTAGATGACATGGATGCTACTTGGG
ATTTATTACAAGAAGAATTTGATGAAGAAATGAAAGAAGACTTAGAGCAA
GAGGTCATTAATTTTAAGGCTAAAGTGGATGAATACGAATTGCAATTATT
ATTAGATGGGCCTCACGATGCCAATAACGCAATTCTAGAGTTACATCCTG
GTGCAGGTGGCACGGAGTCTCAAGATTGGGCTAATATGCTATTTAGAATG
TATCAACGTTATTGTGAGAAGAAAGGCTTTAAAGTTGAAACTGTTGATTA
TCTACCTGGGGATGAAGCGGGGATTAAAAGTGTAACATTGCTCATCAAAG
GGCATAATGCTTATGGTTATTTAAAAGCTGAAAAAGGTGTACACCGACTA
GTACGAATTTCTCCATTTGATTCATCAGGACGTCGTCATACATCATTTGC
ATCATGCGACGTTATTCCAGATTTTAATAATGATGAAATAGAGATTGAAA
TCAATCCGGATGATATTACAGTTGATACATTCAGAGCTTCTGGTGCAGGT
GGTCAGCATATTAACAAAACTGAATCGGCAATACGAATTACCCACCACCC
CTCAGGTATAGTTGTTAATAACCAAAATGAACGTTCTCAAATTAAAAACC
GTGAAGCAGCTATGAAAATGTTAAAGTCTAAATTATATCAATTAAAATTG
GAAGAGCAGGCACGTGAAATGGCTGAAATTCGTGGCGAACAAAAAGAAAT
CGGCTGGGGAAGCCAAATTAGATCATATGTTTTCCATCCATACTCAATGG
TGAAAGATCATCGTACGAACGAAGAAACAGGTAAGGTTGATGCAGTGATG
GATGGAGACATTGGACCATTTATCGAATCATATTTAAGACAGACAATGTC
GCACGATTAATATATATTTTAAAACCGAGGCTCTAAAAGGGCGTCGGTTT
TTGGTTTTTTTAAAGGTAGCTAAATAAATTGTAAATTAGATTTTGGAATA TGATTTGTTTATGAA
>HGS028, SEQ ID NO: 36
MELSEIKRNIDKYNQDLTQIRGSLDLENKETNIQEYEEMMAEPNFWDNQT
KAQDIIDKNNALKAIVNGYXTLQAEVDDMDATWDLLQEEFDEEMKEDLEQ
EVINFKAKVDEYELQLLLDGPHDANNAILELHPGAGGTESQDWANMLFRM
YQRYCEKKGFKVETVDYLPGDEAGIKSVTLLIKGHNAYGYLKAEKGVHRL
VRISPFDSSGRRMTSFASCDVIPDFNNDEIEIEINPDDITVDTFRASGAG
GQHINKTESAIRITHHPSGIVVNNQNERSQIKNREAAMKMLKSKLYQLKL
EEQAREMAEIRGEQKEIGWGSQIRSYVFHPYSMVKDHRTNEETGKVDAVM
DGDIGPFIESYLRQTMSHD >HGS030, SEQ ID NO: 37, Tmk, thymidylate
kinase AATAACTGAAAATATGATAGAATTGGTAAATGAATATCTGGAAACTG- GAA
TGATAGTTGAAGGAATTAAAAATAATAAAATTTTAGTTGAGGATGAATAA
AATGTCAGCTTTTATAACTTTTGAGGGCCCAGAAGGCTCTGGAAAAACAA
CTGTAATTAATGAAGTTTACCATAGATTAGTAAAAGATTATGATGTCATT
ATGACTAGAGAACCAGGTGGTGTTCCTACTGGTGAAGAAATACGTAAAAT
TGTATTAGAAGGCAATGATATGGACATTAGAACTGAAGCAATGTTATTTG
CTGCATCTAGAAGAGAACATCTTGTATTAAAGGTCATACCAGCTTTAAAA
GAAGGTAAGGTTGTGTTGTGTGATCGCTATATCGATAGTTCATTAGCTTA
TCAAGGTTATGCTAGAGGGATTGGCGTTGAAGAAGTAAGAGCATTAAACG
AATTTGCAATAAATGGATTATATCCAGACTTGACGATTTATTTAAATGTT
AGTGCTGAAGTAGGTCGCGAACGTATTATTAAAAATTCAAGAGATCAAAA
TAGATTAGATCAAGAAGATTTAAAGTTTCACGAAAAAGTAATTGAAGGTT
ACCAAGAAATCATTCATAATGAATCACAACGGTTCAAAAGCGTTAATGCA
GATCAACCTCTTGAAAATGTTGTTGAAGACACGTATCAAACTATCATCAA
ATATTTAGAAAAGATATGATATAATTGTTAGAAGAGGTGTTATAAAATGA
AAATGATTATAGCGATCGTACAAGATCAAGATAGTCAGGAACTTGCAGAT
CAACTTGTTAAAAATAACTTTAGAGCAACAAAATTGGCAA >HGS030, SEQ ID NO: 38,
tmk, thymidylate kinase MSAFITFEGPEGSGKTTVINEVYHRLVKDYD-
VIMTREPGGVPTGEEIRKI VLEGNDMDIRTEAMLFAASRREHLVLKVIPALKEGKVV-
LCDRYIDSSLAY QGYARGIGVEEVRALNEFAINGLYPDLTIYLNVSAEVGRERIIKN- SRDQN
RLDQEDLKFHEKVIEGYQEIIHNESQRFKSVNADQPLENVVEDTYQTIIK YLEKI
>HGS031, SEQ ID NO: 39, PyrH, uridylate kinase
AATGTTGCTTTATTAAAATGTAAATCATTCTAATAAAACGACAACTGTG- T
CTTCTTTACTTGTATATGTTACATATATTCACGATAGAGAGGATAAGAAA
ATGGCTCAAATTTCTAAATATAAACGTGTAGTTTTGAAACTAAGTGGTGA
AGCGTTAGCTGGAGAAAAAGGATTTGGCATAAATCCAGTAATTATTAAAA
GTGTTGCTGAGCAAGTGGCTGAAGTTGCTAAAATGGACTGTGAAATCGCA
GTAATCGTTGGTGGCGGAAACATTTGGAGAGGTAAAACAGGTAGTGACTT
AGGTATGGACCGTGGAACTGCTGATTACATGGGTATGCTTGCAACTGTAA
TGAATGCCTTAGCATTACAAGATAGTTTAGAACAATTGGATTGTGATACA
CGAGTATTAACATCTATTGAAATGAAGCAAGTGGCTGAACCTTATATTCG
TCGTCGTGCAATTAGACACTTAGAAAAGAAACGCGTAGTTATTTTTGCTG
CAGGTATTGGAAACCCATACTTCTCTACAGATACTACAGCGGCATTACGT
GCTGCAGAAGTTGAAGCAGATGTTATTTTAATGGGCAAAAATAATGTAGA
TGGTGTATATTCTGCAGATCCTAAAGTAAACAAAGATGCGGTAAAATATG
AACATTTAACGCATATTCAAATGCTTCAAGAAGGTTTACAAGTAATGGAT
TCAACAGCATCCTCATTCTGTATGGATAATAACATTCCGTTAACTGTTTT
CTCTATTATGGAAGAAGGAAATATTAAACGTGCTGTTATGGGTGAAAAGA
TAGGTACGTTAATTACAAAATAAATTTAGAGGTGTAAAATAATGAGTGAC
ATTATTAATGAAACTAAATCAAGAATGCAAAAATCAATCGAAAGCTTATC
ACGTGAATTAGCTAACATCAGTG >HGS031, SEQ ID NO: 40, pyrH, uridylate
kinase MAQISKYKRVVLKLSGEALAGEKGFGINPVIIKSVAEQVAEVAKMDCEI- A
VIVGGGNIWRGKTGSDLGMDRGTADYMGMLATVMNALALQDSLEQLDCDT
RVLTSIEMKQVAEPYIRRRAIRHLEKKRVVIFAAGIGNPYFSTDTTAALR
AAEVEADVILMGKNNVDGVYSADPKVNKDAVKYEHLTHIQMLQEGLQVML
STASSFCMDNNIPLTVFSIMEEGNIKRAVMGEKIGTLITK >HGS032, SEQ ID NO: 41
GATAGCATCCATGTATAGTGATAGTATTTACAACAATTATTATAATACTA
TTTAGTTAAGTAGAGAAATAGTTAAACATTTGAAAGTGTGGTTTAATGGA
ATGTCAGCAATAGGAACAGTTTTTAAAGAACATGTAAAGAACTTTTATTT
AATTCAAAGACTGGCTCAGTTTCAAGTTAAAATTATCAATCATAGTAACT
ATTTAGGTGTGGCTTGGGAATTAATTAACCCTGTTATGCAAATTATGGTT
TACTGGATGGTTTTTGGATTAGGAATAAGAAGTAATGCACCAATTCATGG
TGTACCTTTTGTTTATTGGTTATTGGTTGGTATCAGTATGTGGTTCTTCA
TCAACCAAGGTATTTTAGAAGGTACTAAAGCAATTACACAAAAGTTTAAT
CAAGTATCGAAAATGAACTTCCCGTTATCGATAATACCGACATATATTGT
GACAAGTAGATTTTATGGACATTTAGGCTTACTTTTACTTGTGATAATTG
CATGTATGTTTACTGGTATTTATCCATCAATACATATCATTCAATTATTG
ATATATGTACCGTTTTGTTTTTTCTTAACTGCCTCGGTGACGTTATTAAC
ATCAACACTCGGTGTGTTAGTTAGAGATACACAAATGTTAATGCAAGCAA
TATTAAGAATATTATTTTACTTTTCACCAATTTTGTGGCTACCAAAGAAC
CATGGTATCAGTGGTTTAATTCATGAAATGATGAAATATAATCCAGTTTA
CTTTATTGCTGAATCATACCGTGCAGCAATTTTATATCACGAATGGTATT
TCATGGATCATTGGAAATTAATGTTATACAATTTCGGTATTGTTGCCATT
TTCTTTGCAATTGGTGCGTACTTACACATGAAATATAGAGATCAATTTGC
AGACTTCTTGTAATATATTTATATGACGAAACCCCGCTAACCATTAATAA
ATGGAAGTGGGGTTCATTTTTGTTTATAATTTAAGTAAATAACATATTAA GTTGGTGTATTAT
>HGS032, SEQ ID NO: 42
MSAIGTVFKEHVKNFYLIQRLAQFQVKIINHSNYLGVAWELINPVMQIMV
YWMVFGLGIRSNAPIHGVPFVYWLLVGISMWFFINQGILEGTKAITQKFN
QVSKMNFPLSIIPTYIVTSRFYGHLGLLLLVIIACMFTGIYPSIHIIQLL
IYVPFCFFLTASVTLLTSTLGVLVRDTQMLMQAILRILFYFSPILWLPKN
HGISGLIHEMMKYNPVYFIAESYRAAILYHEWYFMDHWKLMLYNFGIVAI
FFAIGAYLHMKYRDQFADFL >HGS033, SEQ ID NO: 43
TAACAAAATCTTCTATACACTTTACAACAGGTTTTAAAATTTAACAACTG
TTGAGTAGTATATTATAATCTAGATAAATGTGAATAAGGAAGGTCTACAA
ATGAACGTTTCGGTAAACATTAAAAATGTAACAAAAGAATATCGTATTTA
TCGTACAAATAAAGAACGTATGAAAGATGCGCTCATTCCCAAACATAAAA
ACAAAACATTTTTCGCTTTAGATGACATTAGTTTAAAAGCATATGAAGGT
GACGTCATAGGGCTTGTTGGCATCAATGGTTCCGGCAAATCAACGTTGAG
CAATATCATTGGCGGTTCTTTGTCGCCTACTGTTGGCAAAGTGGATCGTA
ATGGTGAAGTCAGCGTTATCGCAATTAGTGCTGGCTTGAGTGGACAACTT
ACAGGGATTGAAAATATCGAATTTAAAATGTTATGTATGGGCTTTAAGCG
AAAAGAAATTAAAGCGATGACACCTAAGATTATTGAATTTAGTGAACTTG
GTGAGTTTATTTATCAACCAGTTAAAAAGTATTCAAGTGGTATGCGTGCA
AAACTTGGTTTTTCAATTAATATCACAGTTAATCCAGATATCTTAGTCAT
TGACGAAGCTTTATCTGTAGGTGACCAAACTTTTGCACAAAAATGTTTAG
ATAAAATTTACGAGTTTAAAGAGCAAAACAAAACCATCTTTTTCGTTAGT
CATAACTTAGGACAAGTGAGACAATTTTGTACTAAGATTGCTTGGATTGA
AGGCGGAAAGTTAAAAGATTACGGTGAACTTGATGATGTATTACCTAAAT
ATGAAGCTTTCCTTAACGATTTTAAAAAGAAATCCAAAGCCGAACAAAAA
GAATTTAGAAACAAACTCGATGAGTCCCGCTTCGTTATTAAATAAACCGA
AAAAACCGAGAATCTCCATTTAAGGATTTCCTCGGTTTTATTTTTGTCAT
CATGATTATTTCGCCTTTTTTATTTTTCTTTTTGCTTTGGCTATT >HGS033, SEQ ID
NO: 44 MNVSVNIKNVTKEYRIYRTNKERMKDALIPKHKNKTFFALDDISLKAYEG
DVIGLVGINGSGKSTLSNIIGGSLSPTVGKVDRNGEVSVIAISAGLSGQL
TGIENIEFKMLCMGFKRKEIKAMTPKIIEFSELGEFIYQPVKKYSSGMRA
KLGFSINITVNPDILVIDEALSVGDQTFAQKCLDKIYEFKEQNKTIFFVS
HNLGQVRQFCTKIAWIEGGKLKDYGELDDVLPKYEAFLNDFKKKSKAEQK EFRNKLDESRFVIK
>HGS034, SEQ ID NO: 45
ATAAGGTGAAGACACATAAAACAATATATCTTAGTAAGCATGCAACACTC
TTTTTTGTTTATTCATAACAACAAAAAAGAATTAAAGGAGGAGTCTTATT
ATGGCTCGATTCAGAGGTTCAAACTGGAAAAAATCTCGTCGTTTAGGTAT
CTCTTTAAGCGGTACTGGTAAAGAATTAGAAAAACGTCCTTACGCACCAG
GACAACATGGTCCAAACCAACGTAAAAAATTATCAGAATATGGTTTACAA
TTACGTGAAAAACAAAAATTACGTTACTTATATGGAATGACTGAAAGACA
ATTCCGTAACACATTTGACATCGCTGGTAAAAAATTCGGTGTACACGGTG
AAAACTTCATGATCTTATTAGCAAGTCGTTTAGACGCTGTTGTTTATTCA
TTAGGTTTAGCTCGTACTCGTCGTCAAGCACGTCAATTAGTTAACCACGG
TCATATCTTAGTAGATGGTAAACGTGTTGATATTCCATCTTATTCTGTTA
AACCTGGTCAAACAATTTCAGTTCGTGAAAAATCTCAAAAATTAAACATC
ATCGTTGAATCAGTTGAAATCAACAATTTCGTACCTGAGTACTTAAACTT
TGATGCTGACAGCTTAACTGGTACTTTCGTACGTTTACCAGAACGTAGCG
AATTACCTGCTGAAATTAACGAACAATTAATCCGTTGAGTACTACTCAAG
ATAATACGGTCAATACCAACACCCACAATTGTGGGTGT >HGS034, SEQ ID NO: 46
MARFRGSNWKKSRRLGISLSGTGKELEKRPYAPGQHGPNQRXKLSEYGLQ
LREKQKLRYLYGMTERQFRNTFDIAGKKFGVHGENFMILLASRLDAVVYS
LGLARTRRQARQLVNHGHILVDGKRVDIPSYSVKPGQTISVREKSQKLNI
IVESVEINNFVPEYLNFDADSLTGTFVRLPERSELPAEINEQLIR >HGS036, SEQ ID
NO: 47 TGTTGATTGCACCTGCTTCAGTCATTGCTATAACTATTTTAATTTTTAAT
TTAACCGGTGATGCACTAAGAGATAGATTGCTGAAACAACGGGGTGAATA
TGATGAGTCTCATTGATATACAAAATTTAACAATAAAGAATACTAGTGAG
AAATCTCTTATTAAAGGGATTGATTTGAAAATTTTTAGTCAACAGATTAA
TGCCTTGATTGGAGAGAGCGGCGCTGGAAAAAGTTTGATTGCTAAAGCTT
TACTTGAATATTTACCATTTGATTTAAGCTGCACGTATGATTCGTACCAA
TTTGATGGGGAAAATGTTAGTAGATTGAGTCAATATTATGGTCATACAAT
TGGCTATATTTCTCAAAATTATGCAGAAAGTTTTAACGACCATACTAAAT
TAGGTAAACAGTTAACTGCGATTTATCGTAAGCATTATAAAGGTAGTAAA
GAAGAGGCTTTGTCCAAAGTTGATAAGGCTTTGTCGTGGGTTAATTTACA
AAGCAAAGATATATTAAATAAATATAGTTTCCAACTTTCTGGGGGCCAAC
TTGAACGCGTATACATAGCAAGCGTTCTCATGTTGGAGCCTAAATTAATC
ATTGCAGACGAACCAGTTGCATCATTGGATGCTTTGAACGGTAATCAAGT
GATGGATTTATTACAGCATATTGTATTAGAACATGGTCAAACATTATTTA
TTATCACACATAACTTAAGTCATGTATTGAAATATTGTCAGTACATTTAT
GTTTTAAAAGAAGGTCAAATCATTGAACGAGGTAATATTAATCATTTCAA
GTATGAGCATTTGCATCCGTATACTGAACGTCTAATTAAATATAGAACAC
AATTAAAGAGGGATTACTATGATTGAGTTAAAACATGTGACTTTTGGTTA
TAATAAAAAGCAGATGGTGCTACAAGATATCAATATTACTATACCTGATG
GAGAAAATGTTGGTATTTTAGGCGAAAGTG >HGS036, SEQ ID NO: 48
MMSLIDIQNLTIKNTSEKSLIKGIDLKIFSQQINALIGESGAGKSLIAKA
LLEYLPFDLSCTYDSYQFDGENVSRLSQYYGHTIGYISQNYAESFNDHTK
LGKQLTAIYRXHYXGSKEEALSKVDKALSWVNLQSKDILNKYSFQLSGGQ
LERVYIASVLMLEPKLIIADEPVASLDALNGNQVNDLLQHIVLEHGQTLF
IITHNLSHVLKYCQYIYVLKEGQIIERGNINHFKYEHLHPYTERLIKYRT QLKRDYYD
>HGS040, SEQ ID NO: 49
GATGATATTTTAATTACAGAAAATGGTTGTCAAGTCTTTACTAAATGCAC
AAAAGACCTTATAGTTTTAACATAAGCGTGTAAAATGAGGAGGAAACTGA
ATGATTTCGGTTAATGATTTTAAAACAGGTTTAACAATTTCTGTTGATAA
CGCTATTTGGAAAGTTATAGACTTCCAACATGTAAAGCCTGGTAAAGGTT
CAGCATTCGTTCGTTCAAAATTACGTAATTTAAGAACTGGTGCAATTCAA
GAGAAAACGTTTAGAGCTGGTGAAAAAGTTGAACCAGCAATGATTGAAAA
TCGTCGCATGCAATATTTATATGCTGACGGRGATAATCATGTATTTATGG
ATAATGAAAGCTTTGAACAAACAGAACTTTCAAGTGATTACTTAAAAGAA
GAATTGAATTACTTAAAAGAAGGTATGGAAGTACAAATTCAAACATACGA
AGGTGAAACTATCGGTGTTGAATTACCTAAAACTGTTGAATTAACAGTAA
CTGAAACAGAACCTGGTATTAAAGGTGATACTGCAACTGGTGCCACTAAA
TCGGCAACTGTTGAAACTGGTTATACATTAAATGTACCTTTATTTGTAAA
CGAAGGTGACGTTTTAATTATCAACACTGGTGATGGAAGCTACATTTCAA
GAGGATAATCTCTAATTTGTTAACAAATAGCTTGTATTCACTATACTGAT
TTAACGTAAGANATTCTAAATAAGTCTCATAAAGCTATTGCCTAAAATGA TTATAGGTTA
>HGS040, SEQ ID NO: 50
MISVNDFKTGLTISVDNAIWKVIDFQHKPGKGSAFVRSKLRNLRTGAIQE
KTFRAGEKVEPAMIENRRMQYLYADGDNHVFMDNESFEQTELSSDYLKEE
LNYLKEGMEVQIQTYEGETIGVELPKTVELTVTETEPGIKGDTATGATKS
ATVETGYTLNVPLFVNEGDVLIINTGDGSYISRG >168153/168339, SEQ ID NO:
51, (operon comprising ORFs for five polypeptides listed below)
TTAGGATGTAAGAAAGTTCCAGTGCAAGAAATCCATGAAACACAATATTC
AATTAGTACATGGCAACATAAAGTTCCATTTGGTGTGTGGTGGGAAACGT
TACAACAAGAACATCGCTTGCCATGGACTACTGAGACAAGACAAGAAGCG
CCATTTATTACAATGTGTCATGGTGATACAGAACAATATTTGTATACAAA
AGATTTAGGCGAAGCACATTTTCAAGTATGGGAAAAGGTTGTCGCAAGTT
ATAGTGGTTGTTGTTCTGTAGAGAGAATTGCACAAGGTACATATCCTTGT
CTTTCTCAACAAGATGTACTCATGAAGTATCAGCCATTGAGTTATAAGGA
AATTGAAGCGGTTGTTCATAAAGGGGAAACTGTGCCAGCAGGTGTGACAC
GCTTTAATATTTCAGGACGATGTCTTAATCTTCAAGTACCACTGGCATTA
CTTAAACAAGATGATGATGTTGAACAATGCGCAATTGGAAGCAGTTTTTA
GCAGATAAGTTTGCCAATATGAGATGCTATACTGAAAAAGTATACTTGGT
GGAGCAATAGTTTTACTGTGATGTTGAGGGAAATATGATGATTTAGCGTA
TTGATAGCGAAAATATAATAAAACAATATAGTGTGGAGAACTTTTGATAT
TTTATAAATATTGAAGTTCTCCATTTTTGTATTTTGCATATAAAAATTAA
ATAAAATAAGGTATATTAAGGTAAAGTATAAATTTTAAATAAATGGGGAA
TGAGTATGAGCTCAATTATAGGAAAAATAGCAATTTGGATAGGCATCGTA
GCTCAAATATATTTTAGTGTCGTTTTTGTTAGGATGATATCTATTAATAT
TGCTGGAGGATCTGATTACGAAACAATTTTTTTATTAGGATTAATATTGG
CTCTTTTCACTGTTTTACCAACCATCTTTACTGCGATTTATATGGAAAGT
TACTCTGTAATCGGAGGTGCACTTTTTATTGTTTATGCTATTATTGCACT
GTGTTTATATAATTTCCTTTCGTCAATTTTATGGCTGATTGGTGGTATTT
TGCTGATTTGGAATAAATACTCAAAAGATGAATCGACAGACGAAAATGAA
AAAGTTGATATTGAAAGTACAGAGAATCAATTTGAATCTAAAGATAAAAT
CACTAAAGAATAAAGAGAATATTTAAGGTAAAGTATAAATTTTAAATAAA
TGGGGAATAGACATGGAAAAAAATGTAGAAAAATCATTCATAAAGATAGG
TTTATATTTTCAAATAGCTTATATAGTACTCATGGCTATAACTTTATGTG
GGTTTGTAATTTGCTATGGACTAATTTTCGGCCTTTTCTATTTATTATCA
GGTAGCAGAGCTGATTATTTAATAGTAACAATAGTTATATCGGCAATAAT
TTCTATATTTGTAATTATACTTTCAATCGTACCTGTCATCGTATTGGCAT
CTGACTTATTTAAAGAAAGGATTTCAAAAGGTGTCATATTAATTGTATTG
GCTATTATCGCTTTAGTATTATGCAACTTTGTATCTGCAATACTCTGGTT
TGTTTCAGCCATATCTATTTTAGGTAGAAAAAAATTAGTAGCTGCAGCAG
ATACTACCACTATTCAAAAAAGTAAAGGGAACGCAAATCAAGCATCACAT
AAAGACACGTGTAAAAAGGAACTTGATAGTCAAGACATGATGGAACATCC
TGAGGTTAAAAATCCCACGACTAAAAACCTTGAAGGATTTAACGAAGAAA
TACATAAAGATGAAGCTACAACTAAAGTTGTCAGTGATAACACGGAACCG
CCTATTGAATCAAAAGACCATGTCTCGAAAAAAGATTGATGACAAACTAA
TCGAGAGACTTAAAAAAATAATATTCAACATAAGAACTTTTAAAACGACA
TTTAAACGCATTGCCAATCACTAATGGTAGTGCGTTTAACTATACCTTAA
ATATCTGAATATTTTGTTAAATGGAGCTACCTTTGTTGTACTATTCAAAT
GAAGAGGAGTAAAATGTAATTAAAGGAAAGAAATTTGAGGAGTGATCTTT
ATGACAAACAACAAAGTAGCATTAGTAACTGGCGGAGCACAAGGGATTGG
TTTTAAAATTGCAGAACGTTTAGTGGAAGATGGTTTCAAAGTAGCAGTTG
TTGATTTCAATGAAGAAGGGGCAAAAGCAGCTGCACTTAAATTATCAAGT
GATGGTACAAAAGCTATTGCTATCAAAGCAGATGTATCAAACCGTGATGA
TGTATTTAACGCATAAGACAAACTGCCGCGCAATTTGGCGATTTCCATGT
CATGGTTAACAATGCCGGCCTTGGACCAACAACACCAATCGATACAATTA
CTGAAGAACAGTTTAAAACAGTATATGGCGTGAACGTTGCAGGTGTGCTA
TGGGGTATTCAAGCCGCACATGAACAATTTAAAAAATTCAATCATGGCGG
TAAAATTATCAATGCAACATCTCAAGCAGGCGTTGAGGGTAACCCAGGCT
TGTCTTTATATTGCAGTACAAAATTCGCAGTGCGAGGTTTAACACAAGTA
GCCGCACAAGATTTAGCGTCTGAAGGTATTACTGTGAATGCATTCGCACC
TGGTATCGTTCAAACACCAATGATGGAAAGTATCGCAGTGGCAACAGCCG
AAGAAGCAGGTAAACCTGAAGCATGGGGTTGGGAACAATTTACAAGTCAG
ATTGCTTTGGGCAGAGTTTCTCAACCAGAAGATGTTTCAAATGTAGTGAG
CTTCTTAGCTGGTAAAGACTCTGATTACATTACTGGACAAACAATTATTG
TAGATGGTGGTATGAGATTCCGTTAATAATCATCCACTAATGATAAATAA
ATCCTTATTGTTAAGTTTAATCACTTAGCAGTAAGGATTTTTTAGTGCAC
TTAGAAGGGAGTGTATTGGTAGAAAATTAATAAGCGAAGTTCTTAAGTGA
GTTATGATGTCACAGTCTAATGCATCAGTTGAAAGCATTATTAGTATTAA
CACACCCAAGATATTATAAAACATCACAAAAACACCACTATCTAATTTAT
CTCAATAAAAATTCACAAAGTTATCTCATTTTATTTTTATAAATAAAAAA
TATCGATAAAAAGCTTACAATACTTTATGTTTTTATGATATATTTTTAAT
GTATAAATGAGGTGGAAGATTTGGAAAGAGTTTTGATAACTGGTGGGGCT
GGTTTTATTGGGTCGCATTTAGTAGATGATTTACAACAAGATTATGATGT
TTATGTTCTAGATAACTATAGAACAGGTAAACGAGAAAATATTAAAAGTT
TGGCTGACGATCATGTGTTTGAATTAGATATTCGTGAATATGATGCAGTT
GAACAAATCATGAAGACATATCAATTTGATTATGTTATTCATTTAGCAGC
ATTAGTTAGTGTTGCTGAGTCGGTTGAGAAACCTATCTTATCTCAAGAAA
TAAACGTCGTAGCAACATTAAGATTGTTAGAAATCATTAAAAAATATAAT
AATCATATAAAACGTTTTATCTTTGCTTCGTCAGCAGCTGTTTATGGTGA
TCTTCCTGATTTGCCTAAAAGTGATCAATCATTAATCTTACCATTATCAC
CATATGCAATAGATAAATATTACGGCGAACGGACGACATTAAATTATTGT
TCGTTATATAACATACCAACAGCGGTTGTTAAATTTTTTAATGTATTTGG
GCCAAGACAGGATCCTAAGTCACAATATTCAGGTGTGATTTCAAAGATGT
TCGATTCATTTGAGCATAACAAGCCATTTACATTTTTTGGTGACGGACTG
CAAACTAGAGATTTTGTATATGTATATGATGTTGTTCAATCTGTACGCTT
AATTATGGAACACAAAGATGCAATTGGACACGGTTATAACATTGGTACAG
GCACTTTTACTAATTTATTAGAGGTTTATCGTATTATTGGTGAATTATAT
GGAAAATCAGTCGAGCATGAATTTAAAGAAGCACGAAAAGGAGATATTAA
GCATTCTTATGCAGATATTTCTAACTTAAAGGCATTAGGATTTGTTCCTA
AATATACAGTAGAAACAGGTTTAAAGGATTACTTTAATTTTGAGGTAGAT
AATATTGAAGAAGTTACAGCTAAAGAAGTGGAAATGTCGTGAAAATGACA
TTGAAGCTGTCCATAATAATAAGGGTTATGCCTATCAAAGAAAATTAGAC
AAACTAGAAGAAGTGAGAAAAAGCTATTACCCAATTAAACGTGCGATTGA
CTTAATTTTAAGCATTGTTTTATTATTTTTAACTTTACCGATTATGGTTA
TATTCGCCATTGCTATCGTCATAGATTCGCCAGGAAACCCTATTTATAGT
CAGGTTAGAGTTGGGAAGATGGGTAAATTAATTAAAATATACAAATTACG
TTCGATGTGCAAAAACGCAGAGAAAAACGGTGCGCAATGGGCTGATAAAG
ATGATGATCGTATAACAAATGTCGGGAAGTTTATTCGTAAAACACGCATT
GATGAATTACCACAACTAATTAATGTTGTTAAAGGGGAAATGAGTTTTAT
TGGACCACGCCCGGAACGTCCGGAATTTGTAGAATTATTTAGTTCAGAAG
TGATAGGTTTCGAGCAAAGATGTCTTGTTACACCAGGGTTAACAGGACTT
GCGCAAATTCAAGGTGGATATGACTTAACACCGCAACAAAAACTGAAATA
TGACATGAAATATATACATAAAGGTAGTTTAATGATGGAACTATATATAT
CAATTAGAACATTGATGGTTGTTATTACAGGGGAAGGCTCAAGGTAGTCT
TAATTTACTTAATAAGTTCAAATAAAAGTTATATTTTAAAGATTGTGACC
AATTGTTACAGTATAACGAGGAATCCCTTGAGACAGTATCAAATGGCATT
AAGAAATATGTGCCATCATTGATTTGCATGGCTATAAATACTATTCATCT
GATGAGATAGCCATGTTAAGAAATTGAAAGTATAGCATTAAAGGGGTTTG
TAACAGTTGAAAATTATATATTGTATTACTAAAGCAGACAATGGTGGTGC
ACAAACACATCTCATTCAACTCGCCAACCATTTTTGCGTACACAATGATG
TTTATGTCATTGTAGGCAATCATGGACCAATGATTGAACAACTAGATGCA
AGAGTTAATGTAATTATTATCGAACATTTAGTAGGTCCAATTGACTTTAA
ACAAGATATTTTAGCTGTCAAAGTGTTAGCACAGTTATTCTCGAAAATTA
AACCTGATGTTATCCATTTACATTCTTCCAAAGCTGGAACGGTCGGACGA
ATTGCGAAGTTCATTTCGAAATCGAAAGACACACGTATAGTTTTTACTGC
ACATGGATGGGCTTTTACAGAGGGTGTTAAACCAGCTAAAAAATTTCTAT
ATTTAGTTATCGAAAAATTAATGTCACTTATTACAGATAGCATTATTTGT
GTTTCAGATTTCGATAAACAGTTAGCGTTAAAATATCGATTTAATCGATT
GAAATTAACCACAATACATAATGGTATTGCAGATGTTCCCGCTGTTAAGC
AAACGCTAAAAAGCCAATCACATAACAATATTGGCGAAGTAGTTGGAATG
TTGCCTAATAAACAAGATTTACAGATTAATGCCCCGACAAAGCATCAATT
TGTTATGATTGCAAGATTTGCTTATCCAAAATTGCCACAAAATCTAATCG
CGGCAATAGAGATATTGAAATTACATAACAGTAATCATGCGCATTTTACA
TTTATAGGCGATGGACCTACATTAAATGATTGTCAGCAACAAGTTGTACA
AGCTGGGTTAGAAAATGATGTCACATTTTTGGGCAATGTCATTAATGCGA
GTCATTTATTATCACAATACGATACGTTTATTTTAATAAGTAAGCATGAA
GGTTTGCCAATTAGCATTATAGAAGCTATGGCTACAGGTTTGCCTGTTAT
AGCCAGTCATGTTGGCGGTATTTCAGAATTAGTAGCTGATAATGGTATAT
GTATGATGAACAACCAACCCGAAACTATTGCTAAAGTCCTGGAAAAATAT
TTAATAGACAGTGATTACATCAAAATGAGTAATCAATCTAGAAAACGTTA
TTTAGAATGTTTTACTGAGGAGAAAATGATTAAAGAAGTGGAAGACGTTT
ATAATGGAAAATCAACACAATAGTAAATTACTAACATTGTTACTTATCGG
TTTAGCGGTTTTTATTCAGCAATCTTCGGTTATTGCCGGTGTGAATGTTT
CTATAGCTGACTTTATCACATTACTAATATTAGTTTATTTACTGTTTTTC
GCTAACCATTTATTAAAGGCAAATCATTTTTTACAGTTTTTCATTATTTT
GTATACATATCGTATGATTATTACGCTTTGTTTGCTATTTTTTGATGATT
TGATATTTATTACGGTTAAGGAAGTTCTTGCATCTACAGTTAAATATGCA
TTTGTAGTCATTTATTTCTATTTAGGGATGATCATCTTTAAGTTAGGTAA
TAGCAAAAAAGTGATCGTTACCTCTTATATTATAAGCAGTGTGACTATAG
GTCTATTTTGTATTATAGCTGGTTTGAACAAGTCCCCTTTACTAATGAAA
TTGTTATATTTTGATGAAATACGTTCAAAAGGATTAATGAATGACCCTAA
CTATTTCGCGATGACACAGATTATTACATTGGTACTTGCTTACAAGTATA
TTCATAATTACATATTCAAGGTCCTTGCATGTGGTATTTTGCTATGGTCT
TTAACTACAACGGGGTCTAAGACTGCGTTTATCATATTAATCGTCTTAGC
CATTTATTTCTTTATTAAAAAGTTATTTAGTAGAAATGCGGTAAGTGTTG
TGAGTATGTCAGTGATTATGCTGATATTACTTTGTTTTACCTTTTATAAT
ATCAACTACTATTTATTCCAATTAAGCGACCTTGATGCCTTACCGTCATT
AGATCGAATGGCGTCTATTTTTGAAGAGGGCTTTGCATCATTAAATGATA
GTGGGTCTGAGCGAAGTGTTGTATGGATAAATGCCATTTCAGTAATTAAA
TATACACTAGGTTTTGGTGTCGGATTAGTGGATTATGTACATATTGGCTC
GCAAATTAATGGTATTTTACTTGTTGCCCATAATACATATTTGCAGATCT
TTGCGGAATGGGGCATTTTATTCGGTGCATTATTTATCATATTTATGCTT
TATTTACTGTTTGAATTATTTAGATTTAACATTTCTGGGAAAAATGTAAC
AGCAATTGTTGTAATGTTGACGATGCTGATTTACTTTTTAACAGTATCAT
TTAATAACTCAAGATATGTCGCTTTTATTTTAGGAATTATCGTCTTTATT
GTTCAATATGAAAAGATGGAAAGGGATCGTAATGAAGAGTGATTCACTAA
AAGAAAATATTATTTATCAAGGGCTATACCAATTGATTAGAACGATGACA
CCACTGATTACAATACCCATTATTTCACGTGCATTTGGTCCCAGTGGTGT
GGGTATTGTTTCATTTTCTTTCAATATCGTGCAATACTTTTTGATGATTG
CAAGTGTTGGCGTTCAGTTATATTTTAATAGAGTTATCGCGAAGTCCGTT
AACGACAAACGGCAATTGTCACAGCAGTTTTGGGATATCTTTGTCAGTAA
ATTATTTTTAGCGTTAACAGTTTTTGCGATGTATATGGTCGTAATTACTA
TATTTATTGATGATTACTATCTTATTTTCCTACTACAAGGAATCTATATT
ATAGGTGCAGCACTCGATATTTCATGGTTTTATGCTGGAACTGAAAAGTT
TAAAATTCCTAGCCTCAGTAATATTGTTGCGTCTGGTATTGTATTAAGTG
TAGTTGTTATTTTTGTCAAAGATCAATCAGATTTATCATTGTATGTATTT
ACTATTGCTATTGTGACGGTATTAAACCAATTACCTTTGTTTATCTATTT
AAAACGATACATTAGCTTTGTTTCGGTTAATTGGATACACGTCTGGCAAT
TGTTTCGTTCGTCATTAGCATACTTATTACCAAATGGACAGCTCAACTTA
TATACTAGTATTTCTTGCGTTGTTCTTGGTTTAGTAGGTACATACCAACA
AGTTGGTATCTTTTCTAACGCATTTAATATTTTAACGGTCGCAATCATAA
TGATTAATACATTTGATCTTGTAATGATTCCGCGTATTACCAAAATGTCT
ATCCAGCAATCACATAGTTTAACTAAAACGTTAGCTAATAATATGAATAT
TCAATTGATATTAACAATACCTATGGTCTTTGGTTTAATTGCAATTATGC
CATCATTTTATTTATGGTTCTTTGGTGAGGAATTCGCATCAACTGTCCCA
TTGATGACCATTTTAGCGATACTTGTATTAATCATTCCTTTAAATATGTT
GATAAGCAGGCAATATTTATTAATAGTGAATAAAATAAGATTATATAATG
CGTCAATTACTATTGGTGCAGTGATAAACCTAGTATTATGTATTATTTTG
ATATATTTTTATGGAATTTACGGTGCTGCTATTGCGCGTTTAATTACAGA
GTTTTTCTTGCTCATTTGGCGATTTATTGATATTACTAAAATCAATGTGA
AGTTGAATATTGTAAGTACGATTCAATGTGTCATTGCTGCTGTTATGATG
TTTATTGTGCTTGGTGTGGTCAATCATTATTTGCCCCCTACAATGTACGC
TACGCTGCTATTAATTGCGATTGGTATAGTAGTTTATCTTTTATTAATGA
TGACTATGAAAAATCAATACGTATGGCAAATATTGAGGCATCTTCGACAT
AAAACAATTTAAGTACCGGTAATGCTATACTTTAGAAAATTAAGATTAAG
AAGAAAAGGCAATTTCTTATTGAAAAATGGAAGTTGTCTTTTTTAATTCT
CTTTAAAAGCGGGAAACAAAAGCAGTTAAATGCCTTTTTGCATTCAATAT
TAAATATTATATCAATTTCGAATATTTAAATTTTATATAATTGGATATAA
CAAATAAATAATAATTATTGCAAAACACACCCAAAATTAATTATTATAAA
AGTATATTCATAAAAGGAGGAATATACTTATGGCATTTAAATTACCAAAT
TTACCATATGCATATGATGCATTGGAACCATATATAGATCAAAGAACAAT
GGAGTTTCATCACGACAAACATCACAATACGTACGTGACGAAATTAAACG
CAACAGTTGAAGGAACAGAGTTAGAGCATCAATCACTAGCGGATATGATT
GCTAACTTAGACAAGGTACCGGAAGCGATGGGGTACCGAGCTCGAATTCG
TAATCATGTCATAGCTGTTTCCTGTG >168153_3, SEQ ID NO: 52
GTGGAAGATTTGGAAAGAGTTTTGATAACTGGTGGGGCTGGTTTTATTGG
GTCGCATTTAGTAGATGATTTACAACAAGATTATGATGTTTATGTTCTAG
ATAACTATAGAACAGGTAAACGAGAAAATATTAAAAGTTTGGCTGACGAT
CATGTGTTTGAATTAGATATTCGTGAATATGATGCAGTTGAACAAATCAT
GAAGACATATCAATTTGATTATGTTATTCATTTAGCAGCATTAGTTAGTG
TTGCTGAGTCGGTTGAGAAACCTATCTTATCTCAAGAAATAAACGTCGTA
GCAACATTAAGATTGTTAGAAATCATTAAAAAATATAATAATCATATAAA
ACGTTTTATCTTTGCTTCGTCAGCAGCTGTTTATGGTGATCTTCCTGATT
TGCCTAAAAGTGATCAATCATTAATCTTACCATTATCACCATATGCAATA
GATAAATATTACGGCGAACGGACGACATTAAATTATTGTTCGTTATATAA
CATACCAACAGCGGTTGTTAAATTTTTTAATGTATTTGGGCCAAGACAGG
ATCCTAAGTCACAATATTCAGGTGTGATTTCAAAGATGTTCGATTCATTT
GAGCATAACAAGCCATTTACATTTTTTGGTGACGGACTGCAAACTAGAGA
TTTTGTATATGTATATGATGTTGTTCAATCTGTACGCTTAATTATGGAAC
ACAAAGATGCAATTGGACACGGTTATAACATTGGTACAGGCACTTTTACT
AATTTATTAGAGGTTTATCGTATTATTGGTGAATTATATGGAAAATCAGT
CGAGCATGAATTTAAAGAAGCACGAAAAGGAGATATTAAGCATTCTTATG
CAGATATTTCTAACTTAAAGGCATTAGGATTTGTTCCTAAATATACAGTA
GAAACAGGTTTAAAGGATTACTTTTTTCAGGTAGATAATATTGAAGAAGT
TACAGCTAAAGAAGTGGAAATGTCGTGA >168153_3, SEQ ID NO: 53
VEDLERVLITGGAGFIGSHLVDDLQQDYDVYVLDNYRTGKRENIKSLADD
HVFELDIREYDAVEQIMKTYQFDYVIHLAALVSVAESVEKPILSQEINVV
ATLRLLEIIKKYNNHIRRPIFASSAAVYGDLPDLPKSDQSLILPLSPYAI
DKYYGERTTLNYCSLYNIPTAVVKFFNVFGPRQDPKSQYSGVISKMFDSF
EHNKPFTFFGDGLQTRDFVYVYDVVQSVRLIMEHKDAIGHGYNIGTGTFT
NLLEVYRIIGELYGKSVEHEFKEARKGDIKHSYADISNLKALGFVPKYTV
ETGLKDYFNFEVDNIEEVTAKEVEMS >168153_2, SEQ ID NO: 54
ATGGTTATATTCGCCATTGCTATCGTCATAGATTCGCCAGGAAACCCTAT
TTATAGTCAGGTTAGAGTTGGGAAGATGGGTAAATTAATTAAAATATACA
AATTACGTTCGATGTGCAAAAACGCAGAGAAAAACGGTGCGCAATGGGCT
GATAAAGATGATGATCGTATAACAAATGTCGGGAAGTTTATTCGTAAAAC
ACGCATTGATGAATTACCACAACTAATTAATGTTGTTAAAGGGGAAATGA
GTTTTATTGGACCACGCCCGGAACGTCCGGAATTTGTAGAATTATTTAGT
TCAGAAGTGATAGGTTTCGAGCAAAGATGTCTTGTTACACCAGGGTTAAC
AGGACTTGCGCAAATTCAAGGTGGATATGACTTAACACCGCAACAAAAAC
TGAAATATGACATGAAATATATACATAAAGGTAGTTTAATGATGGAACTA
TATATATCAATTAGAACATTGATGGTTGTTATTACAGGGGAAGGCTCAAG GTAG
>168153_2, SEQ ID NO: 55 LDKLEEVRKSYYPIKRAIDLILSI-
VLLFLTLPIMVIFAIAIVIDSPGNPI YSQVRVGKMGKLIKIYKLRSMCKNAEKNGAQ-
WADKDDDRITNVGKFIRKT RIDELPQLINVVKGEMSFIGPRPERPEFVELFSSEVIG-
FEQRCLVTPGLT GLAQIQGGYDLTPQQKLKYDMKYIHKGSLMMELYISIRTLMVVIT- GEGSR
>168153_1, SEQ ID NO: 56
ATGATTGAACAACTAGATGCAAGAGTTAATGTAATTATTATCGAACATTT
AGTAGGTCCAATTGACTTTAAACAAGATATTTTAGCTGTCAAAGTGTTAG
CACAGTTATTCTCGAAAATTAAACCTGATGTTATCCATTTACATTCTTCC
AAAGCTGGAACGGTCGGACGAATTGCGAAGTTCATTTCGAAATCGAAAGA
CACACGTATAGTTTTTACTGCACATGGATGGGCTTTTACAGAGGGTGTTA
AACCAGCTAAAAAATTTCTATATTTAGTTATCGAAAAATTAATGTCACTT
ATTACAGATAGCATTATTTGTGTTTCAGATTTCGATAAACAGTTAGCGTT
AAAATATCGATTTAATCGATTGAAATTAACCACAATACATAATGGTATTG
CAGATGTTCCCGCTGTTAAGCAAACGCTAAAAAGCCAATCACATAACAAT
ATTGGCGAAGTAGTTGGAATGTTGCCTAATAAACAAGATTTACAGATTAA
TGCCCCGACAAAGCATCAATTTGTTATGATTGCAAGATTTGCTTATCCAA
AATTGCCACAAAATCTAATCGCGGCAATAGAGATATTGAAATTACATAAC
AGTAATCATGCGCATTTTACATTTATAGGCGATGGACCTACATTAAATGA
TTGTCAGCAACAAGTTGTACAAGCTGGGTTAGAAAATGATGTCACATTTT
TGGGCAATGTCATTAATGCGAGTCATTTATTATCACAATACGATACGTTT
ATTTTAATAAGTAAGCATGAAGGTTTGCCAATTAGCATTATAGAAGCTAT
GGCTACAGGTTTGCCTGTTATAGCCAGTCATGTTGGCGGTATTTCAGAAT
TAGTAGCTGATAATGGTATATGTATGATGAACAACCAACCCGAAACTATT
GCTAAAGTCCTGGAAAAATATTTAATAGACAGTGATTACATCAAAATGAG
TAATCAATCTAGAAAACGTTATTTAGAATGTTTTACTGAGGAGAAAATGA
TTAAAGAAGTGGAAGACGTTTATAATGGAAAATCAACACAATAG >168153_1, SEQ ID
NO: 57 LKIIYCITKADNGGAQTHLIQLANHFCVHNDVYVIVGN- HGPMIEQLDARV
NVIIIEHLVGPIDFKQDILAVKVLAQLFSKIKPDVIHLHSSKAGT- VGRIA
XFISKSKDTRIVFTAHGWAFTEGVKPAKKFLYLVIEKLMSLITDSIICVS
DFDKQLALKYRFNRLKLTTIHNGIADVPAVKQTLKSQSHNNIGEVVGMLP
NKQDLQINAPTKHQFVMIARFAYPKLPQNLIAAIEILKLHNSNHAHFTFI
GDGPTLNDCQQQVVQAGLENDVTFLGNVINASHLLSQYDTFILISKHEGL
PISIIEAMATGLPVIASHVGGISELVADNGICMMNNQPETIAKVLEKYLI
DSDYIKMSNQSRKRYLECFTEEKMIKEVEDVYNGKSTQ >168339_1, SEQ ID NO: 58,
(ORF overlaps the 3' end of 168153_1 by 20 nucleotides)
ATGGAAAATCAACACAATAGTAAATTACTAACATTGTTACTTATCGGTTT
AGCGGTTTTTATTCAGCAATCTTCGGTTATTGCCGGTGTGAATGTTTCTA
TAGCTGACTTTATCACATTACTAATATTAGTTTATTTACTGTTTTTCGCT
AACCATTTATTAAAGGCAAATCATTTTTTACAGTTTTTCATTATTTTGTA
TACATATCGTATGATTATTACGCTTTGTTTGCTATTTTTTGATGATTTGA
TATTTATTACGGTTAAGGAAGTTCTTGCATCTACAGTTAAATATGCATTT
GTAGTCATTTATTTCTATTTAGGGATGATCATCTTTAAGTTAGGTAATAG
CAAAAAAGTGATCGTTACCTCTTATATTATAAGCAGTGTGACTATAGGTC
TATTTTGTATTATAGCTGGTTTGAACAAGTCCCCTTTACTAATGAAATTG
TTATATTTTGATGAAATACGTTCAAAAGGATTAATGAATGACCCTAACTA
TTTCGCGATGACACAGATTATTACATTGGTACTTGCTTACAAGTATATTC
ATAATTACATATTCAAGGTCCTTGCATGTGGTATTTTGCTATGGTCTTTA
ACTACAACGGGGTCTAAGACTGCGTTTATCATATTAATCGTCTTAGCCAT
TTATTTCTTTATTAAAAAGTTATTTAGTAGAAATGCGGTAAGTGTTGTGA
GTATGTCAGTGATTATGCTGATATTACTTTGTTTTACCTTTTATAATATC
AACTACTATTTATTCCAATTAAGCGACCTTGATGCCTTACCGTCATTAGA
TCGAATGGCGTCTATTTTTGAAGAGGGCTTTGCATCATTAAATGATAGTG
GGTCTGAGCGAAGTGTTGTATGGATAAATGCCATTTCAGTAATTAAATAT
ACACTAGGTTTTGGTGTCGGATTAGTGGATTATGTACATATTGGCTCGCA
AATTAATGGTATTTTACTTGTTGCCCATAATACATATTTGCAGATCTTTG
CGGAATGGGGCATTTTATTCGGTGCATTATTTATCATATTTATGCTTTAT
TTACTGTTTGAATTATTTAGATTTAACATTTCTGGGAAAAATGTAACAGC
AATTGTTGTAATGTTGACGATGCTGATTTACTTTTTAACAGTATCATTTA
ATAACTCAAGATATGTCGCTTTTATTTTAGGAATTATCGTCTTTATTGTT
CAATATGAAAAGATGGAAAGGGATCGTAATGAAGAGTGA >168339_1, SEQ ID NO: 59
MENQHNSKLLTLLLIGLAVFIQQSSVIAGVNVSIADFITLLILVYLLFFA
NHLLKANHFLQFFIILYTYRMIITLCLLFFDDLIFITVKEVLASTVKYAF
VVIYFYLGMIIFKLGNSKKVIVTSYIISSVTIGLFCIIAGLNXSPLLMKL
LYFDEIRSKGLMNDPNYFAMTQIITLVLAYKYIHNYIFKVLACGILLWSL
TTTGSKTAFIILIVLAIYFFIKKLFSRNAVSVVSMSVIMLILLCFTFYNI
NYYLFQLSDLDALPSLDRMASIFEEGFASLNDSGSERSVVWINAISVIKY
TLGFGVGLVDYVHIGSQINGILLVAHNTYLQIFAEWGILFGALFIIFMLY
LLFELFRFNISGKNVTAIVVMLTMLIYFLTVSFNNSRYVAFILGIIVFIV QYEKMERDRNEE
>168339_2, SEQ ID NO: 60, (ORF overlaps the 3' end of 168339_1
by 35 nucleotides)
ATGAAAAGATGGAAAGGGATCGTAATGAAGAGTGATTCACTAAAAGAAAA
TATTATTTATCAAGGGCTATACCAATTGATTAGAACGATGACACCACTGA
TTACAATACCCATTATTTCACGTGCATTTGGTCCCAGTGGTGTGGGTATT
GTTTCATTTTCTTTCAATATCGTGCAATACTTTTTGATGATTGCAAGTGT
TGGCGTTCAGTTATATTTTAATAGAGTTATCGCGAAGTCCGTTAACGACA
AACGGCAATTGTCACAGCAGTTTTGGGATATCTTTGTCAGTAAATTATTT
TTAGCGTTAACAGTTTTTGCGATGTATATGGTCGTAATTACTATATTTAT
TGATGATTACTATCTTATTTTCCTACTACAAGGAATCTATATTATAGGTG
CAGCACTCGATATTTCATGGTTTTATGCTGGAACTGAAAAGTTTAAAATT
CCTAGCCTCAGTAATATTGTTGCGTCTGGTATTGTATTAAGTGTAGTTGT
TATTTTTGTCAAAGATCAATCAGATTTATCATTGTATGTATTTACTATTG
CTATTGTGACGGTATTAAACCAATTACCTTTGTTTATCTATTTAAAACGA
TACATTAGCTTTGTTTCGGTTAATTGGATACACGTCTGGCAATTGTTTCG
TTCGTCATTAGCATACTTATTACCAAATGGACAGCTCAACTTATATACTA
GTATTTCTTGCGTTGTTCTTGGTTTAGTAGGTACATACCAACAAGTTGGT
ATCTTTTCTAACGCATTTAATATTTTAACGGTCGCAATCATAATGATTAA
TACATTTGATCTTGTAATGATTCCGCGTATTACCAAAATGTCTATCCAGC
AATCACATAGTTTAACTAAAACGTTAGCTAATAATATGAATATTCAATPG
ATATTAACAATACCTATGGTCTTTGGTTTAATTGCAATTATGCCATCATT
TTATTTATGGTTCTTTGGTGAGGAATTCGCATCAACTGPCCCATTGATGA
CCATTTTAGCGATACTTGTATTAATCATTCCTTTAAATATGTTGATAAGC
AGGCAATATTTATTAATAGTGAATAAAATAAGATTATATAATGCGTCAAT
TACTATTGGTGCAGTGATAAACCTAGTATTATGTATTATTTTGATATATT
TTTATGGAATTTACGGTGCTGCTATTGCGCGTTTAATTACAGAGTTTTTC
TTGCTCATTTGGCGATTTATTGATATTACTAAAATCAATGTGAAGTTGAA
TATTGTAAGTACGATTCAATGTGTCATTGCTGCTGTTATGATGTTTATTG
TGCTTGGTGTGGTCAATCATTATTTGCCCCCTACAATGTACGCTACGCTG
CTATTAATTGCGATTGGTATAGTAGTTTATCTTTTATTAATGATGACTAT
GAAAAATCAATACGTATGGCAAATATTGAGGCATCTTCGACATAAAACAA TTTAA
>168339_2, SEQ ID NO: 61
MKSDSLKENIIYQGLYQLIRTMTPLITIPIISRAFGPSGVGIVSFSFNIV
QYFLMIASVGVQLYFNRVIAKSVNDKRQLSQQFWDIFVSKLFLALTVFAM
YMVVITIFIDDYYLIFLLQGIYIIGAALDISWFYAGTEKFKIPSLSNIVA
SGIVLSVVVIFVKDQSDLSLYVFTIAIVTVLNQLPLFIYLKRYISFVSVN
WIHVWQLFRSSLAYLLPNGQLNLYTSISCVVLGLVGTYQQVGIFSNAFNI
LTVAIIMINTFDLVMIPRITKMSIQQSHSLTKTLANNNNIQLILTIPMVF
GLIAIMPSFYLWFFGEEFASTVPLMTILAILVLIIPLNMLISRQYLLIVN
XIRLYNASITIGAVINLVLCIILIYFYGIYGAAIARLITEFFLLIWRFID
ITKINVKLNIVSTIQCVIAAVMMFIVLGVVNHYLPPTMYATLLLIAIGIV
VYLLLMMTMKNQYVWQILRHLRHKTI
[0062] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, DNA and genomic DNA obtained by cloning or produced
synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known as
the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
[0063] The present invention further encompasses nucleic acid
molecules of the present invention that are chemically synthesized,
or reproduced as peptide nucleic acids (PNA), or according to other
methods known in the art. The use of PNAs would serve as the
preferred form if the nucleic acid molecules of the invention are
incorporated onto a solid support, or gene chip. For the purposes
of the present invention, a peptide nucleic acid (PNA) is a
polyamide type of DNA analog and the monomeric units for adenine,
guanine, thymine and cytosine are available commercially
(Perceptive Biosystems). Certain components of DNA, such as
phosphorus, phosphorus oxides, or deoxyribose derivatives, are not
present in PNAS. As disclosed by P. E. Nielsen, M. Egholm, R. H.
Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O.
Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver,
R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,
666 (1993),
[0064] PNAs bind specifically and tightly to complementary DNA
strands and are not degraded by nucleases. In fact, a PNA binds
more strongly to DNA than does DNA itself. This is probably because
there is no electrostatic repulsion between the two strands, and
also the polyamide backbone is more flexible. Because of this,
PNA/DNA duplexes bind under a wider range of stringency conditions
than DNA/DNA duplexes, making it easier to perform multiplex
hybridization. Smaller probes can be used than with DNA due to the
strong binding. In addition, it is more likely that single base
mismatches can be determined with PNA/DNA hybridization because a
single mismatch in a PNA/DNA 15-mer lowers the melting point
(T.sup..sub.m) by 8.degree.-20.degree. C., vs. 4.degree.-16.degree.
C. for the DNA/DNA 15-mer duplex. Also, the absence of charge
groups in PNA means that hybridization can be done at low ionic
strengths and reduce possible interference by salt during the
analysis.
[0065] By "isolated" polynucleotide sequence is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. This includes segments of DNA comprising the S. aureus
polynucleotides of the present invention isolated from the native
chromosome. These fragments include both isolated fragments
consisting only of S. aureus DNA and fragments comprising
heterologous sequences such as vector sequences or other foreign
DNA. For example, recombinant DNA molecules contained in a vector
are considered isolated for the purposes of the present invention
which may be partially or substantially purified. Further examples
of isolated DNA molecules include recombinant DNA molecules
introduced and maintained in heterologous host cells or purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
DNA molecules of the present invention. Isolated nucleic acid
molecules according to the present invention further include such
molecules produced synthetically which may be partially or
substantially purified the excluded RNA or heterologous DNA.
Isolated nucleic acid molecules at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative
to herelogous (Staphylococcus or other) (DNA or RNA) or relative to
all materials and compounds other than the carrier solution. The
term "isolated" does not refer to genomic or cDNA libraries, whole
cell mRNA preparations, genomic DNA digests (including those gel
separated by electrophoresis), whole chromosome or sheared whole
cell genomic DNA preparations or other compositions where the art
demonstrates no distinguishing features of the polynucleotides
sequences of the present invention.
[0066] In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode a S. aureus
polypeptides and peptides of the present invention (e.g.,
polypeptides of Table 1). That is, all possible DNA sequences that
encode the S. aureus polypeptides of the present invention. This
includes the genetic code and species-specific codon preferences
known in the art. Thus, it would be routine for one skilled in the
art to generate the degenerate variants described above, for
instance, to optimize codon expression for a particular host (e.g.,
change codons in the bacteria mRNA to those preferred by a
mammalian or other bacterial host such as E. coli).
[0067] The invention further provides isolated nucleic acid
molecules having the nucleotide sequence shown in Table 1 or a
nucleic acid molecule having a sequence complementary to one of the
above sequences. Such isolated molecules, particularly DNA
molecules, are useful as probes for gene mapping and for
identifying S. aureus in a biological sample, for instance, by PCR
or hybridization analysis (e.g., including but not limited to,
Northern blot analysis). In specific embodiments, the
polynucleotides of the present invention are less than 300 kb, 200
kb, 100 kb, 50 kb, 10,kb, 7.5 kb, 5 kb, 2.5 kb, and 1 kb. In
another embodiment, the polynucleotides comprising the coding
sequence for polypeptides of the present invention do not contain
genomic flanking gene sequences or contain only genomic flanking
gene sequences having regulatory control sequences for the said
polynucleotides.
[0068] The present invention is further directed to nucleic acid
molecules encoding portions or fragments of the nucleotide
sequences described herein. Uses for the polynucleotide fragments
of the present invention include probes, primers, molecular weight
markers and for expressing the polypeptide fragments of the present
invention. Fragments include portions of the nucleotide sequences
of Table 1, at least 10 contiguous nucleotides in length selected
from any two integers, one of which representing a 5' nucleotide
position and a second of which representing a 3' nucleotide
position, where the first nucleotide for each nucleotide sequence
in Table 1 is position 1. That is, every combination of a 5' and 3'
nucleotide position that a fragment at least 10 contiguous
nucleotides in length could occupy is included in the invention as
an individual species. "At least" means a fragment may be 10
contiguous nucleotide bases in length or any integer between 10 and
the length of an entire nucleotide sequence minus 1. Therefore,
included in the invention are contiguous fragments specified by any
5' and 3' nucleotide base positions of a nucleotide sequences of
Table 1 wherein the contiguous fragment is any integer between 10
and the length of an entire nucleotide sequence minus 1.
[0069] The polynucleotide fragment specified by 5' and 3' positions
can be immediately envisaged using the clone description and are
therefore not individually listed solely for the purpose of not
unnecessarily lengthening the specifications.
[0070] Although it is particularly pointed out that each of the
above described species may be included in or excluded from the
present invention. The above species of polynucleotides fragments
of the present invention may alternatively be described by the
formula "a to b"; where "a" equals the 5' nucleotide position and
"b" equals 3' nucleotide position of the polynucleotide fragment,
where "a" equals an integer between 1 and the number of nucleotides
of the polynucleotide sequence of the present invention minus 10,
where "b" equals an integer between 10 and the number of
nucleotides of the polynucleotide sequence of the present
invention; and where "a" is an integer smaller then "b" by at least
10.
[0071] Again, it is particularly pointed out that each species of
the above formula may be specifically included in, or excluded
from, the present invention. Further, the invention includes
polynucleotides comprising sub-genuses of fragments specified by
size, in nucleotides, rather than by nucleotide positions. The
invention includes any fragment size, in contiguous nucleotides,
selected from integers between 10 and the length of an entire
nucleotide sequence minus 1. Preferred size of contiguous
nucleotide fragments include at least 20 nucleotides, at least 30
nucleotides, at least 40 nucleotides, at least 50 nucleotides, at
least 60 nucleotides, at least 70 nucleotides, at least 80
nucleotides, at least 90 nucleotides, at least 100 nucleotides, at
least 125 nucleotides, at least 150 nucleotides, at least 175
nucleotides, at least 200 nucleotides, at least 250 nucleotides, at
least 300 nucleotides, at least 350 nucleotides, at least 400
nucleotides, at least 450 nucleotides, at least 500 nucleotides, at
least 550 nucleotides, at least 600 nucleotides, at least 650
nucleotides, at least 700 nucleotides, at least 750 nucleotides, at
least 800 nucleotides, at least 850 nucleotides, at least 900
nucleotides, at least 950 nucleotides, at least 1000 nucleotides,
at least 1050 nucleotides, at least 1100 nucleotides, and at least
1150 nucleotides. Other preferred sizes of contiguous
polynucleotide fragments, which may be useful as diagnostic probes
and primers, include fragment sizes representing each integer
between 50-300. Larger fragments are also useful according to the
present invention corresponding to most, if not all, of the
polynucleotide sequences of the sequence listing or Table 1. The
preferred sizes are, of course, meant to exemplify not limit to
present invention as all size fragments, representing any integer
between 10 and the length of an entire nucleotide sequence minus 1
of the sequence listing or deposited clones, may be specifically
included from the invention. Additional preferred nucleic acid
fragment of the present invention include nucleic acid molecules
encoding epitope-bearing portions of the polynucleotides (e.g.,
including but not limited to, nucleic acid molecules encoding
epitope-bearing portions of the polynucleotides which are shown in
Table 4).
[0072] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of a
polynucleotide in a nucleic acid molecules of the invention
described above, for instance, nucleotide sequences of Table 1. By
"stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C. Hybridizing polynucleotides are useful as diagnostic
probes and primers as discussed above. Portions of a polynucleotide
which hybridize to a nucleotide sequence in Table 1, which can be
used as probes and primers, may be precisely specified by 5' and 3'
base positions or by size in nucleotide bases as described above or
precisely excluded in the same manner. Preferred hybridizing
polynucleotdies of the present invention are those that, when
labeled and used in a hybridization assay known in the art (e.g.
Southern and Northern blot analysis), display the greatest signal
strength with the polynucleotides of Table 1 regardless of other
heterologous sequences present in equamolar amounts
[0073] The nucleic acid molecules of the present invention, which
encode a S. aureus polypeptide, may include, but are not limited
to, nucleic acid molecules encoding the full length S. aureus
polypeptides of Table 1. Also included in the present invention are
nucleic acids encoding the above full length sequences and further
comprise additional sequences, such as those encoding an added
secretory leader sequence, such as a pre-, or pro- or
prepro-protein sequence. Further included in the present invention
are nucleic acids encoding the above full length sequences and
portions thereof and further comprise additional heterologous amino
acid sequences encoded by nucleic acid sequences from a different
source.
[0074] Also included in the present invention are nucleic acids
encoding the above protein sequences together with additional,
non-coding sequences, including for example, but not limited to
non-coding 5' and 3' sequences. These sequences include
transcribed, non-translated sequences that may play a role in
transcription, and mRNA processing, for example, ribosome binding
and stability of mRNA. Also included in the present invention are
additional coding sequences which provide additional
functionalities.
[0075] Thus, a nucleotide sequence encoding a polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. For instance, hexa-histidine provides for
convenient purification of the fusion protein. See Gentz et al.
(1989) Proc. Natl. Acad. Sci. 86:821-24. The "HA" tag is another
peptide useful for purification which corresponds to an epitope
derived from the influenza hemagglutinin protein. See Wilson et al.
(1984) Cell 37:767. As discussed below, other such fusion proteins
include the S. aureus fused to Fc at the N- or C-terminus.
[0076] Variant and Mutant Polynucleotides
[0077] The present invention further relates to variants of the
nucleic acid molecules which encode portions, analogs or
derivatives of a S. aureus polypeptides of Table 1, and variant
polypeptides thereof including portions, analogs, and derivatives
of the S. aureus polypeptides. Variants may occur naturally, such
as a natural allelic variant. By an "allelic variant" is intended
one of several alternate forms of a gene occupying a given locus on
a chromosome of an organism. See, e.g., B. Lewin, Genes IV (1990).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0078] Such nucleic acid variants include those produced by
nucleotide substitutions, deletions, or additions. The
substitutions, deletions, or additions may involve one or more
nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of a S. aureus protein of the present
invention or portions thereof. Also preferred in this regard are
conservative substitutions.
[0079] Such polypeptide variants include those produced by amino
acid substitutions, deletions or additions. The substitutions,
deletions, or additions may involve one or more residues.
Alterations may produce conservative or non-conservative amino acid
substitutions, deletions, or additions. Especially preferred among
these are silent substitutions, additions and deletions, which do
not alter the properties and activities of a S. aureus protein of
the present invention or portions thereof. Also especially
preferred in this regard are conservative substitutions.
[0080] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of S. aureus polypeptides or peptides by
recombinant techniques.
[0081] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleic acid sequence shown in Table 1. The above nucleic acid
sequences are included irrespective of whether they encode a
polypeptide having S. aureus activity. This is because even where a
particular nucleic acid molecule does not encode a polypeptide
having S. aureus activity, one of skill in the art would still know
how to use the nucleic acid molecule, for instance, as a
hybridization probe or primer. Uses of the nucleic acid molecules
of the present invention that do not encode a polypeptide having S.
aureus activity include, inter alia, isolating an S. aureus gene or
allelic variants thereof from a DNA library, and detecting S.
aureus mRNA expression in biological or environmental samples,
suspected of containing S. aureus by Northern Blot analysis or
PCR.
[0082] For example, one such method involves assaying for the
expression of a polynucleotide encoding S. aureus polypeptides in a
sample from an animal host (e.g, including, but not limited to,
human, bovine, rabbit, porcine, murine, chicken, and/or avian
species). The expression of polynucleotides can be assayed by
detecting the nucleic acids of Table 1. An example of such a method
involves the use of the polymerase chain reaction (PCR) to amplify
and detect Staphylococcus nucleic acid sequences in a biological or
environmental sample.
[0083] The present invention also relates to nucleic acid probes
having all or part of a nucleotide sequence described in Table 1
which are capable of hybridizing under stringent conditions to
Staphylococcus nucleic acids. The invention further relates to a
method of detecting one or more Staphylococcus nucleic acids in a
biological sample obtained from an animal, said one or more nucleic
acids encoding Staphylococcus polypeptides, comprising: (a)
contacting the sample with one or more of the above-described
nucleic acid probes, under conditions such that hybridization
occurs, and (b) detecting hybridization of said one or more probes
to the Staphylococcus nucleic acid present in the biological
sample.
[0084] The invention also includes a kit for analyzing samples for
the presence of members of the Staphylococcus genus in a biological
or environmental sample. In a general embodiment, the kit includes
at least one polynucleotide probe containing a nucleotide sequence
that will specifically hybridize with a S. aureus nucleic acid
molecule of Table 1 and a suitable container. In a specific
embodiment, the kit includes two polynucleotide probes defining an
internal region of the S. aureus nucleic acid molecule of Table 1,
where each probe has one strand containing a 31'mer-end internal to
the region. In a further embodiment, the probes may be useful as
primers for polymerase chain reaction amplification.
[0085] The method(s) provided above may preferrably be applied in a
diagnostic method and/or kits in which S. aureus polynucleotides of
Table 1 are attached to a solid support. In one exemplary method,
the support may be a "gene chip" or a "biological chip" as
described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174.
Further, such a gene chip with S. aureus polynucleotides of Table 1
attached may be used to diagnose S. aureus infection in an animal
host, preferably a human. The US Patents referenced above are
incorporated herein by reference in their entirety.
[0086] The present invention is further directed to nucleic acid
molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99%
identical to the nucleic acid sequence shown in Table 1, which do,
in fact, encode a polypeptide having S. aureus protein activity. By
"a polypeptide having S. aureus activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the S. aureus protein of the invention, as measured in
a particular biological assay suitable for measuring activity of
the specified protein. The biological activity of some of the
polypeptides of the presents invention are listed in Table 1, after
the name of the closest homolog with similar activity. The
biological activities were determined using methods known in the
art for the particular biological activity listed. For the
remaining polypeptides of Table 1, the assays known in the art to
measure the activity of the polypeptides of Table 2, sharing a high
degree of identity, may be used to measure the activity of the
corresponding polypeptides of Table 1.
[0087] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequences shown in Table 1 will encode a polypeptide having
biological activity. In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing the above
described comparison assay. It will be further recognized in the
art that, for such nucleic acid molecules that are not degenerate
variants, a reasonable number will also encode a polypeptide having
biological activity. This is because the skilled artisan is fully
aware of amino acid substitutions that are either less likely or
not likely to significantly effect protein function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino
acid), as further described below.
[0088] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence of
the present invention, it is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the S. aureus polypeptide. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted, inserted, or
substituted with another nucleotide. The query sequence may be an
entire sequence shown in Table 1, the ORF (open reading frame), or
any fragment specified as described herein.
[0089] Other methods of determining and defining whether any
particular nucleic acid molecule or polypeptide is at least 90%,
95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the
presence invention can be done by using known computer programs. A
preferred method for determining the best overall match between a
query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm
of Brutlag et al. See Brutlag et al. (1990) Comp. App. Biosci.
6:237-245. In a sequence alignment the query and subject sequences
are both DNA sequences. An RNA sequence can be compared by first
converting U's to T's. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB
alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=O, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject
nucleotide sequence, whichever is shorter.
2TABLE 2 Closest matching sequence between the polypeptides of the
present invention and sequences in GenSeq and GenBank databases
Smallest Sum Sequence Probability P ID Antigen Accession No. Match
Gene Name High Score (N) GenSeq HGS001 W34207 Streptomyces fabH
homologue (frenolicin gene I pro . . . 285 3.50E-65 HGS001 W55808
Streptomyces roseofulvus frenolicin gene cluster p . . . 285
3.50E-65 HGS002 W20949 H. pylori cytoplasmic protein,
29zp10241orf7. 81 5.10E-12 HGS003 W48300 Staphylococcus aureus Fab
I enoyl-ACP reductase. 1271 1.90E-170 HGS003 W40806 M. bovis InhA
protein. 95 1.00E-29 HGS003 R23793 Stearoyl-ACP-desaturase (from
clone pDES7). 157 1.60E-28 HGS003 R66290 M. tuberculosis inhA gene.
94 7.40E-28 HGS003 R66901 M. tuberculosis InhA. 94 7.40E-28 HGS003
R66292 Mycobacterium bovis InhA. 92 4.70E-19 HGS003 R63900 M. bovis
InhA. 92 4.70E-19 HGS003 W16684 Lawsonia intracellularis
enoyl-(acyl carrier prote . . . 114 1.80E-09 HGS003 W40805 M.
tuberculosis InhA protein. 96 2.60E-09 HGS003 W40807 M. smegmatis
InhA protein, mc2155 inhA-1. 101 9.70E-09 HGS004 W32287
Streptococcus pneumoniae MurA protein. 643 4.00E-89 HGS004 W26786
Streptococcus pneumoniae Mur A-1. 643 4.10E-89 HGS004 W27782
UDP-N-acetylglucosamine 1-carboxyvinyltransferase. 163 1.80E-15
HGS004 W27783 UDP-N-acetylglucosamine 1-carboxyvinyltransferase.
120 1.90E-12 HGS006 W36168 Staphylococcus aureus SP protein. 584
4.30E-78 HGS006 W37468 Staphylococcus aureus RNase P. 581 1.10E-77
HGS007M W27798 Amino acid sequence of a replicative DNA heli case
5524 126e-83.2 HGS007M R29636 pCTD ORF 1. 241 7e-34.3 HGS008 W27814
A malonyl coenzymeA-acyl carrier protein transacyl . . . 365
4.70E-46 HGS008 W19629 Streptomyces venezuelae polyketide synthase.
96 2.30E-19 HGS008 W22602 Tylactone synthase ORF2 protein. 83
2.90E-18 HGS008 W22605 Tylactone synthase ORF5 protein. 95 8.90E-17
HGS008 R44431 eryA region polypeptide module #2. 88 2.30E-14 HGS008
R42452 Enzyme involved in eicosapentaenoic acid (EPA) syn . . . 94
5.30E-14 HGS008 R99462 Biosynthetic enzyme of icosapentaenoic acid
synthase. 94 4.60E-13 HGS008 W37050 S. putrefaciens EPO
biosynthesis gene cluster ORF6 . . . 94 4.60E-13 HGS008 R44432 eryA
region polypeptide module #3. 83 6.20E-13 HGS008 W22607 Platenolide
synthase ORF2 protein. 80 2.20E-12 HGS014 W34454 Racillus subtilis
teichoic acid polymerase. 597 2.70E-87 HGS014 W34455 Racillus
subtilis teichoic acid polymerase. 597 3.10E-87 HGS014 W27744 Amino
acid sequence of techoic acid biosynthesis p . . . 425 2.50E-53
HGS016 W32287 Streptococcus pneumoniae MurA protein. 643 4.00E-89
HGS016 W26786 Streptococcus pneumoniae Mur A-1. 643 4.10E-89 HGS016
W27782 UDP-N-acetylglucosamine 1-carboxyvinyltransferase. 163
1.80E-15 HGS016 W27783 UDP-N-acetylglucosamine
1-carboxyvinyltransferase. 120 1.90E-12 HGS018 R95648 Thermostable
DNA-ligase. 833 3.00E-205 HGS018 R81473 Thermus aquaticus DNA
ligase protein. 428 2.00E-201 HGS018 R15299 Thermostable T.
aquaticus ligase (I). 428 7.40E-199 HGS018 R15694 Thermostable T.
aquaticus ligase (II). 428 4.80E-196 HGS019 P70096
Met-aminopeptidase. 143 2.90E-35 HGS019 R90027 Methionine
aminopeptidase sequence. 138 1.60E-20 HGS022 R12401
Enantioselective amidase of Rhodococcus. 405 4.70E-102 HGS022
R25320 Enantioselective amidase. 405 4.70E-102 HGS022 W14159
Rhodococcus rhodochrous amidase. 352 6.10E-63 HGS022 W17820
Pseudomonas putida amidase. 208 1.20E-62 HGS022 R12400
Enantioselective amidase of Brevibacterium. 353 2.90E-62 HGS022
R24529 Enantioselective amidase. 353 2.90E-62 HGS022 W10882
Comamonas acidovorans derived amidase enzyme. 261 4.00E-61 HGS022
R60155 Comamonas testosteroni NI 1 amidase. 306 5.30E-47 HGS022
R42839 Urea amidolyase. 243 1.40E-31 HGS022 R44504 Urea amide
lyase. 224 8.60E-30 HGS026 W29380 S. pneumoniae peptide releasing
factor RF-1. 593 3.30E-142 HGS028 W29380 S. pneumoniae peptide
releasing factor RF-1. 218 1.70E-49 HGS031 W20646 H. pylori
cytoplasmic protein, 02cp11822orf26. 291 5.70E-47 HGS031 W20147 H.
pylori cytoplasmic protein, 14574201.aa. 75 1.50E-08 HGS033 W20861
H. pylori cell envelope transporter protein, 12ge1 . . . 100
2.30E-18 HGS033 W20101 H. pylori transporter protein 11132778.aa.
100 6.10E-17 HGS033 W25671 hABC3 protein. 111 4.20E-15 HGS033
W46761 Amino acid sequence of human ATP binding cassette . . . 111
4.20E-15 HGS033 W46771 Amino acid sequence of human ATP binding
cassette . . . 111 4.30E-15 HGS033 W42393 Bacillus thermoleovorans
phosphatase (68FY5). 96 1.90E-13 HGS033 W34202 Streptomyces efflux
pump protein (frenolicin gene . . . 92 5.50E-12 HGS033 W55803
Streptomyces roseofulvus frenolicin gene cluster p . . . 92
5.50E-12 HGS033 W20224 H. pylori transporter protein, 22265691.aa.
88 7.40E-12 HGS033 W20668 H. pylori transporter protein
O3ee11215orf29. 88 8.90E-12 HGS036 W20640 H. pylori transporter
protein, 02ce11022orf8. 264 2.20E-33 HGS036 W34202 Streptomyces
efflux pump protein (frenolicin gene . . . 184 1.30E-29 HGS036
W55803 Streptomyces roseofulvus frenolicin gene cluster p . . . 184
1.30E-29 HGS036 W20289 H. pylori transporter protein, 24218968.aa.
201 5.50E-21 HGS036 W20711 H. pylori transporter protein,
05cp11911orf41. 148 2.10E-19 HGS036 W20101 H. pylori transporter
protein 11132778.aa. 164 3.50E-19 HGS036 W20861 H. pylori cell
envelope transporter protein, 12ge1 . . . 164 4.20E-19 HGS036
W20492 H. pylori cell envelope transporter protein 433843 . . . 148
1.60E-18 HGS036 W21019 H. pylori cell envelope transporter protein,
hp5e1 . . . 144 8.30E-16 HGS036 R71091 C. jejuni PEB1A antigen from
ORF3. 136 7.90E-14 168153_3 W01619 Human uridine diphosphate
galactose-4-epimerase. 128 9.80E-29 168153_3 W40383 S. glaucescens
acbD protein. 105 1.10E-15 168153_3 R98529 dTDP-glucose dehydratase
encoded by the acbB gene. 108 4.50E-15 168153_3 R80287 galE gene of
S. lividans gal operon. 88 2.60E-13 168153_3 P70275 Sequence
encoded by S. lividans gal operon galE gene. 86 5.10E-13 168153_3
R41529 S. lividans UDP-4-epimerase. 86 5.10E-13 168153_3 R32195
ADP-L-glycero-D-mannoheptose-6-epimerase protein. 82 3.40E-10
168153_2 W03997 Glucosyl IP-transferase (SpsB protein). 168
8.30E-36 168153_2 W32794 Sphingomonas genus microbe isolated SpsB
protein. 168 8.30E-36 168153_2 W22173 S. thermophilus
exopolysaccharide synthesis operon . . . 141 2.20E-31 168153_2
W14074 S. thermophilus exopolysaccharide biosynthesis enzy . . .
141 2.20E-31 168153_2 P70458 Sequence of gpD encoded by segment of
Xanthomonas . . . 183 2.30E-30 168153_1 W22175 S. thermophilus
exopolysaccharide synthesis operon . . . 141 6.40E-35 168153_1
W14076 S. thermophilus exopolysaccharide biosynthesis enzy . . .
141 9.50E-35 168153_1 W22174 S. thermophilus exopolysaccharide
synthesis operon . . . 162 9.50E-30 168153_1 W14075 S. thermophilus
exopolysaccharide biosynthesis enzy . . . 162 9.50E-30 168339_2
W27736 Putative O-antigen transporter protein. 820 5.70E-11.5
GenBank HGS001 gnl.vertline.PID.vertline.e1183136 similar to
3-oxoacyl-acyl-carrier protein 569 2.20E-129 HGS001
gi.vertline.151943 ORF3; putative [Rhodobacter capsulatus] 404
1.40E-92 HGS001 gi.vertline.2983572 (AE000723)
3-oxoacyl-[acyl-carrier-protei- n 311 5.10E-92 HGS001
gi.vertline.1276662 beta-ketoacyl-acyl carrier protein synthase 292
3.90E-90 HGS001 gi.vertline.2313291 (AE000540) beta-ketoacyl-acyl
carrier protein 269 3.50E-89 HGS001
gnl.vertline.PID.vertline.e1183019 similar to
3-oxoacyl-acyl-carrier protein 373 2.00E-86 HGS001
gi.vertline.1143069 3-ketoacyl carrier protein synthase III 287
3.60E-86 HGS001 gi.vertline.22744 beta-ketoacyl-acyl carrier
protein synthase 292 1.20E-85 HGS001 gi.vertline.311686
3-ketoacyl-acyl carrier protein synthase 322 3.40E-85 HGS001
gi.vertline.145898 beta-ketoacyl-acyl carrier protein synthase 366
7.30E-84 HGS002 gi.vertline.142833 ORF2 [Bacillus subtilis]
>gnl.vertline.PID.vertline.e11851 . . . 215 2.50E-70 HG5002
gnl.vertline.PID.vertline.d1019368 hypothetical protein
[Synechocystis sp.] 235 8.50E-67 HGS002 gi.vertline.2983165
(AE000694) UDP-N-acetylenolpyruvoylgluco . . . 207 1.10E-58 HGS002
gi.vertline.404010 ORF2 [Bacillus licheniformis]
>pir.vertline.I4022 . . . 251 1.10E-50 HGS002
gi.vertline.2688520 (AE001161) UDP-N-acetylmuramate dehydrog . . .
197 1.80E-42 HGS002 gi.vertline.1841789
UDP-N-acetylenolpyruvylglucosamine reduc . . . 249 7.10E-40 HGS002
gi.vertline.2983149 (AE000693) UDP-N-acetoenolpyruvoylglucos . . .
212 3.80E-36 HGS002 gi.vertline.431730
UDP-N-acetylenolpyruvoylglucosamine redu . . . 119 4.50E-22 HGS002
gi.vertline.1573234 UDP-N-acetylenolpyruvoylglucos- amine redu . .
. 139 6.20E-22 HGS002 gi.vertline.290456
UDP-N-acetylpyruvoylglucosamine reductas . . . 123 2.90E-20 HGS003
gnl.vertline.PID.vertline.e1183192 similar to enoyl-acyl-carrier
protein r . . . 743 1.80E-97 HGS003 gi.vertline.142010 Shows 70.2%
similarity and 48.6% identit . . . 519 8.90E-80 HGS003
gnl.vertline.PID.vertline.d1017769 enoyl-[acyl-carrier-protein]
reductase [. . . 482 2.10E-73 HGS003 gi.vertline.2313282 (AE000539)
enoyl-(acyl-carrier-protein) . . . 449 1.70E-71 HGS003
gi.vertline.145851 envM [Escherichia coli] >gi.vertline.587106
enoyl . . . 388 3.70E-71 HGS003 gi.vertline.153955 envM protein
[Salmonella typhimurium] >p . . . 386 2.10E-69 HGS003
gi.vertline.1574591 short chain alcohol dehydrogenase homolo . . .
362 3.10E-68 HGS003 gi.vertline.2983915 (AE000745)
enoyl-[acyl-carrier-protein] . . . 268 1.10E-64 HGS003
gi.vertline.1053075 orf1; similar to E. coli EnvM [Proteus mi . . .
259 2.60E-29 HGS003 gnl.vertline.PID.vertline.e1188732 (AJ003124)
enoyl-ACP reductase [Petunia . . . 154 2.20E-28 HGS004
gnl.vertline.PID.vertline.e276830 UDP-N-acetylglucosamine
1-carboxyvinyltr . . . 1251 2.50E-195 HGS004 gi.vertline.415662
UDP-N-acetylglucosamine 1-carboxyvinyl t . . . 534 1.40E-139 HGS004
gnl.vertline.PID.vertline.d1010850 UDP-N-acetylglucosamine
1-carboxyvinyltr . . . 732 7.50E-138 HGS004 gi.vertline.41344
UDP-N-acetylglucosamine 1-carboxyvinyltr . . . 537 2.90E-137 HGS004
gi.vertline.1574635 UDP-N-acetylglucosamine enolpyruvyl tran . . .
536 4.70E-136 HGS004 gi.vertline.146902 UDP-N-acetylglucosamine
enolpyruvyl tran . . . 509 5.10E-134 HGS004 gi.vertline.2983705
(AE000732) UDP-N-acetylglucosamine 1-car . . . 492 6.20E-121 HGS004
gnl.vertline.PID.vertline.e229797 UDP-N-acetylglucosamine
enolpyruvyl tran . . . 606 3.00E-119 HGS004 gi.vertline.699337
UDP-N-acetyglucosamine 1-carboxyvinyl tr . . . 605 1.10E-118 HGS004
gi.vertline.2313767 (AE000578) UDP-N-acetylglucosamine enolp . . .
440 1.90E-117 HGS005 gi.vertline.143434 Rho Factor [Bacillus
subtilis] 755 1.10E-190 HGS005 gi.vertline.853769 transcriptional
terminator Rho [Bacillus . . . 746 1.80E-189 HGS005
gi.vertline.2983405 (AE000711) transcriptional terminator Rho . . .
580 2.10E-154 HGS005 gi.vertline.454859 The first ATG in the open
reading frame . . . 543 7.90E-150 HGS005 gi.vertline.147607
transcription termination factor [Escheri . . . 592 9.40E-149
HGS005 gi.vertline.49363 ho Factor [Salmonella typhimurium]
>pirl . . . 592 1.70E-148 HGS005
gnl.vertline.PID.vertline.e220353 Rho gene product [Streptomyces
lividans] . . . 575 4.90E-148 HGS005 gi.vertline.1573263
transcription termination factor rho (rho . . . 575 5.40E-147
HGS005 gi.vertline.49365 Rho factor [Neisseria gonorrhoeae]
>pirl . . . 590 1.40E-146 HGS005 gi.vertline.2313666 (AE000569)
transcription termination fact . . . 547 8.10E-146 HGS006
gi.vertline.580904 homologous to E. coli rnpA [Bacillus subt . . .
295 8.10E-37 HGS006 gnl.vertline.PID.vertline- .d1005777 protein
component of ribonuclease P [Bac . . . 293 1.60E-36 HGS006
gnl.vertline.PID.vertline.d1004132 RNaseP C5 subunit [Mycoplasma
capricolum . . . 99 3.60E-22 HGS006 gi.vertline.144147 rnpA
[Buchnera aphidicola] >gi.vertline.2827012 ( . . . 97 3.90E-10
HGS006 gi.vertline.511457 RNase P protein component [Coxiella burn
. . . 117 2.30E-09 HGS007M gnl.vertline.PID.vertline.d1005718
replicative DNA helicase [Bacillus subti . . . 579 6.20E-169
HGS007M gi.vertline.3282821 (AF045058) DnaC replicative helicase
[Ba . . . 536 3.60E-156 HGS007M gnl.vertline.PID.vertline.e321938
helicase [Rhodothermus marinus] 433 1.50E-123 HGS007M
gi.vertline.2335167 (AF006675) DNA helicase [Rhodothermus ma . . .
271 2.90E-109 HGS007M gnl.vertline.PID.vertline.e211889
DNA-replication helicase [Odontella sine . . . 395 1.60E-108
HGS007M gnl.vertline.PID.vertline.e1263993 (AL022118) replicative
DNA helicase DnaB . . . 235 3.20E-103 HGS007M
gnl.vertline.PID.vertline.e244747 gene 40 [Bacteriophage SPP1]
>gi.vertline.529650 . . . 477 4.40E-103 HGS007M
gi.vertline.2983861 (AE000742) replicative DNA helicase [Aqu . . .
244 1.10E-102 HGS007M gi.vertline.2314528 (AE000636) replicative
DNA helicase (dna . . . 246 7.70E-101 HGS007M
gnl.vertline.PID.vertline.d1011167 replicative DNA helicase
[Synechocystis . . . 209 1.50E-100 HGS008 gnl.vertline.PID.vertlin-
e.e1185181 malonyl CoA-acyl carrier protein transac . . . 560
4.30E-90 HGS008 gi.vertline.1502420 malonyl-CoA:Acyl carrier
protein transac . . . 391 1.40E-86 HGS008 gi.vertline.3282803
(AF044668) malonyl CoA-acyl carrier prot . . . 308 2.50E-75 HGS008
gi.vertline.2738154 malonyl-CoA:acyl carrier protein transac . . .
283 3.40E-75 HGS008 gi.vertline.145887 malonyl coenzyme A-acyl
carrier protein . . . 304 6.30E-75 HGS008 gi.vertline.1573113
malonyl coenzyme A-acyl carrier protein . . . 270 7.60E-74 HGS008
gi.vertline.2983416 (AE000712) malonyl-CoA:Acyl carrier prot . . .
213 2.70E-73 HGS008 gi.vertline.840626 transacylase [Bacillus
subtilis] 221 1.20E-66 HGS008 gi.vertline.3150402 (AC004165)
putative malonyl-CoA:Acyl car . . . 235 1.60E-57 HGS008
gnl.vertline.PID.vertline.e1185300 pksC [Bacillus subtilis]
>gnl.vertline.PID.vertline.e11833 . . . 145 4.40E-38 HGS009
gi.vertline.460911 fructose-bisphosphate aldolase [Bacillus . . .
1169 2.10E-154 HGS009 gnl.vertline.PID.vertline.e1251871
fructose-1,6-bisphosphate aldolase type . . . 1121 6.70E-148 HGS009
gnl.vertline.PID.vertline.d1003809 hypothetical protein [Bacillus
subtilis]. . . 467 1.50E-110 HGS009 gi.vertline.2313265 (AE000538)
fructose-bisphosphate aldolas . . . 252 6.40E-91 HGS009
gi.vertline.1673788 (AE000015) Mycoplasma pneumoniae, fructo . . .
238 4.60E-81 HGS009 gi.vertline.1045692 fructose-bisphosphate
aldolase [Mycoplas . . . 226 6.40E-77 HGS009
gnl.vertline.PID.vertline.d101- 6691 Tagatose-bisphosphate aldolase
GatY (EC . . . 279 2.30E-75 HGS009 gi.vertline.599738 unknown
function [Escherichia coli] >pir . . . 274 2.00E-74 HGS009
gi.vertline.1732204 putative aldolase [Vibrio furnissii] 277
5.00E-74 HGS009 gi.vertline.606077 ORF_o286 [Escherichia coli]
>gi.vertline.1789526 . . . 264 1.30E-73 HGS014 gi.vertline.40100
rodC (tag3) polypeptide (AA 1-746) [Baci . . . 597 1.70E-86 HGS014
gnl.vertline.PID.vertline.e1169895 tasA [Streptococcus pneumoniae]
108 4.90E-27 HGS014 gi.vertline.2621425 (AE000822) teichoic acid
biosynthesis pr . . . 142 2.00E-23 HGS014 gi.vertline.2621421
(AE000822) teichoic acid biosynthesis pr . . . 147 5.90E-22 HGS014
gi.vertline.143725 putative [Bacillus subtilis]
>gnl.vertline.PID.vertline.e1 . . . 114 4.60E-19 HGS014
gi.vertline.547513 orf3 [Haemophilus influenzae]
>pir.vertline.S4924 . . . 106 5.60E-14 HGS014
gnl.vertline.PID.vertline.d1027517 (AB009477) 395aa long
hypothetical prote . . . 79 4.20E-12 HGS014 gi.vertline.2072447
EpsJ [Lactococcus lactis cremoris] 106 5.20E-10 HGS014
gi.vertline.915199 ggaB [Bacillus subtilis]
>gnl.vertline.PID.vertline.e11844 . . . 89 8.10E-08 HGS016
gnl.vertline.PID.vertline.e276830 UDP-N-acetylglucosamine
1-carboxyvinyltr . . . 1251 2.50E-195 HGS016 gi.vertline.415662
UDP-N-acetylglucosamine 1-carboxyvinyl t . . . 534 1.40E-139 HGS016
gnl.vertline.PID.vertline.d1010850 UDP-N-acetylglucosamine
1-carboxyvinyltr . . . 732 7.50E-138 HGS016 gi.vertline.41344
UDP-N-acetylglucosamine 1-carboxyvinyltr . . . 537 2.90E-137 HGS016
gi.vertline.11574635 UDP-N-acetylglucosamine enolpyruvyl tran .
.
. 536 4.70E-136 HGS016 gi.vertline.146902 UDP-N-acetylglucosamine
enolpyruvyl tran . . . 509 5.10E-134 HGS016 gi.vertline.2983705
(AE000732) UDP-N-acetylglucosamine 1-car . . . 492 6.20E-121 HGS016
gnl.vertline.PID.vertline.e229797 UDP-N-acetylglucosamine
enolpyruvyl tran . . . 606 3.00E-119 HGS016 gi.vertline.699337
UDP-N-acetyglucosamine 1-carboxyvinyl tr . . . 605 1.10E-118 H0S016
gi.vertline.2313767 (AE000578) UDP-N-acetylglucosamine enolp . . .
440 1.90E-117 HGS018 gnl.vertline.PID.vertline.e1182642 similar to
DNA ligase [Bacillus subtilis . . . 1574 9.60E-287 HGS018
gnl.vertline.PID.vertline.d1017321 DNA ligase [Synechocystis sp.]
>pir.vertline.S744 . . . 830 5.70E-209 HGS018
gi.vertline.1574651 DNA ligase (lig) [Haemophilus influenzae . . .
484 1.30E-204 HGS018 gi.vertline.607820 DNA ligase [Rhodothermus
marinus] >sp.vertline.P4 . . . 833 1.60E-204 HGS018
gi.vertline.155088 DNA ligase [Thermus aquaticus thermophil . . .
428 3.10E-201 HGS018 gi.vertline.609276 DNA ligase [Thermus
scotoductus] >pir.vertline.S5 . . . 436 1.10E-200 HGS018
gi.vertline.2983242 (AE000699) DNA ligase (NAD dependent) [A . . .
724 1.00E-179 HGS018 gi.vertline.49284 DNA ligase [Zymomonas
mobilis] >pir.vertline.S206 . . . 523 1.60E-170 HGS018
gnl.vertline.PID.vertline.e1237759 (AL021287) DNA ligase
[Mycobacterium tub . . . 529 1.80E-161 HGS018
gnl.vertline.PID.vertline.e349403 DNA ligase [Mycobacterium leprae]
527 7.30E-160 HGS019 dbjl.vertline.D86417_12 YflG [Bacillus
subtilis] >gnl.vertline.PID.ver- tline.e11827 . . . 559 8.00E-72
HGS019 gi.vertline.1044986 methionine aminopeptidase [Bacillus subt
. . . 254 4.50E-58 HGS019 gi.vertline.1574578 methionine
aminopeptidase (map) [Haemoph . . . 185 5.10E-56 HGS019
gnl.vertline.PID.vertline.e1172953 (AL008883) methionine
aminopeptidase [My . . . 214 1.10E-51 HGS019 gi.vertline.2982825
(AE000672) methionyl aminopeptidase [Aqu . . . 192 3.70E-48 HGS019
gnl.vertline.PID.vertline.e1253272 (AL021958) methionine
aminopeptidase [My . . . 130 5.20E-48 HGS019 gi.vertline.2687996
(AE001123) methionine aminopeptidase (ma . . . 195 9.00E-48 HGS019
gnl.vertline.PID.vertline.e1254451 methionine aminopeptidase
[Streptomyces . . . 151 2.10E-43 HGS019 gi.vertline.975723
methionine aminopeptidase I [Saccharomyc . . . 294 3.60E-43 HGS019
gi.vertline.2583129 (AC002387) putative methionine aminopept . . .
211 2.10E-41 HGS022 gnl.vertline.PID.vertline.e118- 2648 alternate
gene name: yedB; similar to am . . . 1586 2.80E-212 HGS022
gi.vertline.2589195 (AF008553) Glu-tRNAGln amidotransferase . . .
1436 1.70E-198 HGS022 gnl.vertline.PID.vertline.d1018331 amidase
[Synechocystis sp.] >pir.vertline.S77264.vertline.. . . 867
2.30E-178 HGS022 gi.vertline.2982954 (AE000680) glutamyl-tRNA (Gln)
amidotran . . . 1247 6.50E-176 HGS022 gi.vertline.1224069 amidase
[Moraxella catarrhahis] >sp.vertline.Q490 . . . 522 4.40E-158
HGS022 gi.vertline.2648182 (AE000943) Glu-tRNA amidotransferase, su
. . . 548 1.30E-145 HGS022 gnl.vertline.PID.vertline.e349405
probable amidase [Mycobacterium leprae] 465 6.30E-143 HGS022
gnl.vertline.PID.vertline.e1237756 (AL021287) putative Glu-tRNA-Gln
amidotr . . . 470 1.90E-141 HGS022 gi.vertline.2313964 (AE000594)
amidase [Helicobacter pylori]. . . 550 7.30E-123 HGS022
gi.vertline.2622613 (AE000910) amidase [Methanobacterium the . . .
524 5.80E-116 HGS023 gi.vertline.1354211 PET112-like protein
[Bacillus subtilis] . . . 2291 2.90E-307 HGS023 gi.vertline.2653657
Bacillus subtilis PET112-like protein [B . . . 1313 1.20E-250
HGS023 gi.vertline.2589196 (AF008553) Glu-tRNAGln amidotransferase
. . . 1315 4.20E-250 HGS023 gnl.vertline.PID.vertline.e1182649
similar to pet112-like protein [Bacillus . . . 1346 7.10E-224
HGS023 gi.vertline.2983123 (AE000691) glutamyl-tRNA (Gln) amidotran
. . . 931 2.30E-165 HGS023 gnl.vertline.PID.vertline.d1019042
PET112 [Synechocystis sp.] >pir.vertline.S75850.vertline.S . . .
859 4.10E-161 HGS023 gi.vertline.1224071 unknown [Moraxella
catarrhalis] >splQ490 . . . 323 3.90E-132 HGS023
gi.vertline.2313783 (AE000579) PET112-like protein [Helicoba . . .
664 6.80E-132 HGS023 gi.vertline.2688237 (AE001140) glu-tRNA
amidotransferase, su . . . 318 4.00E-131 HGS023 gi.vertline.1590917
Glu-tRNA amidotransferase (gatB) [Methan . . . 263 8.60E-125 HGS024
gi.vertline.2465557 (AF011545) YedA [Bacillus subtilis]
>gi.vertline.. . . 237 6.30E-27 HGS024
gnl.vertline.PID.vertline.d1011444 hypothetical protein
[Synechocystis sp.]. . . 153 8.60E-22 HGS024 gi.vertline.2648183
(AE000943) Glu-tRNA amidotransferase, su . . . 126 1.80E-21 HGS024
gnl.vertline.PID.vertline.e1237757 (AL021287) putative Glu-tRNA-Gln
amidotr . . . 166 1.80E-17 HGS024 gi.vertline.2984354 (AE000775)
glutamyl-tRNA (Gln) amidotran . . . 102 2.70E-17 HGS024
gnl.vertline.PID.vertline.e349616 hypothetical protein MLCB637.12
[Mycobac . . . 154 7.10E-16 HGS025 gnl.vertline.PID.vertline.d1005-
830 stage V sporulation [Bacillus subtilis] . . . 496 4.90E-69
HGS025 gnl.vertline.PID.vertline.d1011124 peptidyl-tRNA hydrolase
[Synechocystis s . . . 307 2.10E-49 HGS025 gi.vertline.2983032
(AE000685) peptidyl-tRNA hydrolase [Aqui . . . 386 2.20E-49 HGS025
gnl.vertline.PID.vertline.e304565 Pth [Mycobacterium tuberculosis]
>gnl.vertline.PI . . . 266 2.60E-43 HGS025 gi.vertline.1045760
peptidyl-tRNA hydrolase homolog [Mycopla . . . 211 1.40E-39 HGS025
gi.vertline.2314676 (AE000648) peptidyl-tRNA hydrolase (pth) . . .
102 3.30E-39 HGS025 gi.vertline.1674312 (AE000058) Mycoplasma
pneumoniae, peptid . . . 208 9.50E-39 HGS025 gi.vertline.1127571
peptidyl-tRNA hydrolase [Chlamydia trach . . . 187 7.00E-37 HGS025
gi.vertline.1573366 peptidyl-tRNA hydrolase (pth) [Haemophil . . .
201 8.50E-34 HGS025 gi.vertline.581202 peptidyl-tRNA hydrolase
[Escherichia col . . . 186 2.50E-27 HGS026 gi.vertline.853776
peptide chain release factor 1 [Bacillus . . . 889 6.10E-160 HGS026
gnl.vertline.PID.vertline.d1009421 Peptide Termination Factor
[Mycoplasma c . . . 715 1.10E-126 HGS026 gnl.vertline.PID.vertline-
.d1019559 peptide chain release factor [Synechocys . . . 539
2.70E-121 HGS026 gi.vertline.2688096 (AE001130) peptide chain
release factor . . . 627 1.80E-115 HGS026
gnl.vertline.PID.vertline.d1015453 Peptide chain release factor 1
(RF-1) [E . . . 467 3.90E-113 HGS026 gi.vertline.968930 peptide
chain release factor 1 [Escheric . . . 463 1.30E-112 HGS026
gi.vertline.147567 peptide chain release factor 1 [Escheric . . .
467 3.40E-112 HGS026 gi.vertline.154104 release factor 1
[Salmonella typhimurium . . . 460 2.90E-111 HGS026
gi.vertline.1574404 polypeptide chain release factor 1 (prfA . . .
449 1.50E-109 HGS026 gi.vertline.2313158 (AE000529) peptide chain
release factor . . . 576 1.20E-104 HGS028 gi.vertline.2331287
(AF013188) release factor 2 [Bacillus . . . 769 2.50E-173 HGS028
sp.vertline.P28367.vertline.RF2_BACSU PEPTIDE CHAIN RELEASE FACTOR
2 (RF-2) . . . 742 3.00E-157 HGS028 gi.vertline.2984119 (AE000758)
peptide chain release fact . . . 442 2.20E-128 HGS028
gnl.vertline.PID.vertline.e254636 peptide release factor 2
[Bacillus fi . . . 718 2.90E-125 HGS028
pir.vertline.S76448.vertline.S76448 translation releasing factor
RF-2 - S . . . 883 3.30E-116 HGS028
pir.vertline.A64190.vertline.A64190 translation releasing factor
RF-2 - H . . . 444 1.70E-110 HGS028 gi.vertline.154276 peptide
chain release factor 2 [Salmo . . . 444 1.80E-108 HGS028
gi.vertline.2687953 (AE001120) peptide chain release fact . . . 408
3.90E-108 HGS028 gi.vertline.2367172 (AE000372) peptide chain
release fact . . . 437 1.60E-107 HGS028 gi.vertline.147569 peptide
chain release factor 2 [Esche . . . 434 4.00E-107 HGS030
gnl.vertline.PID.vertline.d1005806 unknown [Bacillus subtilis]
>gnl.vertline.PID.vertline.e11 . . . 283 2.60E-64 HGS030
gi.vertline.3176887 (AF065312) thymidylate kinase [Yersinia . . .
124 3.00E-43 HGS030 gi.vertline.2983484 (AE000716) thymidylate
kinase [Aquifex a . . . 272 2.40E-37 HGS030 gi.vertline.1244710
thymidylate kinase [Escherichia coli] >g . . . 136 7.20E-34
HGS030 gi.vertline.2650584 (AE001102) thymidylate kinase (tmk) [Arc
. . . 71 2.60E-30 HGS030 gi.vertline.1045674 thymidylate kinase
[Mycoplasma genitaliu . . . 173 8.20E-28 HGS030 gi.vertline.1673808
(AE000016) Mycoplasma pneumoniae, thymid . . . 171 1.70E-27 HGS030
gi.vertline.1246364 thymidylate:zeocin resistance protein:ND . . .
136 2.20E-27 HGS030 gi.vertline.1246361 thymidine:thymidylate
kinase:zeocin resi . . . 136 4.30E-27 HGS030 gi.vertline.950071
ATP-bind. pyrimidine kinase [Mycoplasma . . . 80 8.70E-21 HGS031
gnl.vertline.PID.vertline.e1185242 uridylate kinase [Bacillus
subtilis] >pi . . . 920 8.40E-123 HGS031
gnl.vertline.PID.vertline.d1019291 uridine monophosphate kinase
[Synechocys . . . 530 1.70E-96 HGS031 gnl.vertline.PID.vertline.e1-
296663 (AL023797) uridylate kinase [Streptomyce . . . 678 2.10E-89
HGS031 gnl.vertline.PID.vertline.e248883 hypothetical protein
MTCY274.14c [Mycoba . . . 416 6.00E-89 HGS031
gnl.vertline.PID.vertline.e32778- 3 uridylate kinase [Mycobacterium
leprae] 403 7.90E-86 HGS031 gi.vertline.473234 uridine
5'-monophosphate (UMP) kinase [E . . . 384 2.10E-72 HGS031
gi.vertline.1552748 uridine 5'-monophosphate (UMP) kinase [E . . .
375 3.60E-71 HGS031 gi.vertline.1574616 mukB suppressor protein
(smbA) [Haemophi . . . 409 3.70E-71 HGS031 gi.vertline.2983290
(AE000703) UMP kinase [Aquifex aeolicus] 452 3.70E-58 HGS031
gi.vertline.1518662 UMP kinase [Chlamydia trachomatis]
>sp.vertline.P . . . 323 9.10E-55 HGS032 gi.vertline.755152
highly hydrophobic integral membrane pro . . . 297 2.40E-81 HGS032
gi.vertline.1235660 RfbA [Myxococcus xanthus]
>sp.vertline.Q50862.vert- line.RFB . . . 173 4.90E-24 HGS032
gnl.vertline.PID.vertline.d10176- 29 ABC transporter [Synechocystis
sp.] >pir . . . 149 1.50E-19 HGS032
gnl.vertline.PID.vertline.d1029275 (AB010294) integral membrane
component o . . . 126 6.40E-19 HGS032 gnl.vertline.PID.vertline.d1-
008332 putative integral membrane component of . . . 125 9.10E-19
HGS032 gnl.vertline.PID.vertline.d1029271 (AB010293) integral
membrane component o . . . 125 9.10E-19 HGS032
gnl.vertline.PID.vertline.d1- 029279 (AB010295) integral membrane
component o . . . 125 9.10E-19 HGS032
gnl.vertline.PID.vertline.d1029264 (AB10150) integral membrane
component o . . . 109 3.00E-15 HGS032 gi.vertline.2983575
(AE000723) ABC transporter (ABC-2 subfam . . . 71 9.60E-13 HGS032
gi.vertline.609595 homologous to kpsM (E.coli), bexB (H.inf . . .
78 2.60E-12 HGS033 gi.vertline.755153 ATP-binding protein [Bacillus
subtilis] . . . 655 9.30E-94 HGS033 gi.vertline.609596 ATP-binding
protein [Serratia marcescens] 387 3.70E-69 HGS033
gi.vertline.765059 ABC-transporter protein [Klebsiella pneu . . .
371 3.70E-69 HGS033 gi.vertline.567183 ATP-binding protein
[Klebsiella pneumoni . . . 367 1 .20E-67 HGS033 gi.vertline.304013
abcA [Aeromonas salmonicida] >pir.vertline.A36918 . . . 294
7.20E-59 HGS033 gnl.vertline.PID.vertline.d1020415 (AB002668) ABC
transport protein [Actino . . . 323 4.00E-57 HGS033
gi.vertline.1123030 CpxA [Actinobacillus pleuropneumoniae] 190
2.40E-56 HGS033 gi.vertline..vertline.3135679 (AF064070) putative
ABC-2 transporter hy . . . 219 2.10E-53 HGS033 gi.vertline.12983576
(AE000723) ABC transporter [Aquifex aeol . . . 294 2.10E-53 HGS033
gi.vertline.1235661 RfbB [Myxococcus xanthus]
>sp.vertline.Q50863.vert- line.RFB . . . 336 6.70E-53 HGS034
gi.vertline.143467 ribosomal protein S4 [Bacillus subtilis]. . .
798 4.50E-106 HGS034 gi.vertline.2314460 (AE000633) ribosomal
protein S4 (rps4) [. . . 322 1.00E-62 HGS034 gi.vertline.2982819
(AE000672) ribosomal protein S04 [Aquife . . . 253 2.00E-62 HGS034
gi.vertline.606231 30S ribosomal subunit protein S4 [Escher . . .
292 2.40E-58 HGS034 gnl.vertline.PID.vertline.e1234848 (AJ223236)
ribosomal protein S4 [Salmone . . . 292 6.10E-58 HGS034
gi.vertline.1573812 ribosomal protein S4 (rpS4) [Haemophilus . . .
292 1.60E-57 HGS034 gi.vertline.639791 ribosomal protein S4
[Mycoplasma pneumon . . . 260 1.90E-56 HGS034 gi.vertline.1046011
ribosomal protein S4 [Mycoplasma genital . . . 245 2.10E-54 HGS034
gnl.vertline.PID.vertline.e316061 RpsD [Mycobacterium tuberculosis]
>gnl.vertline.P . . . 270 1.40E-52 HGS034 gi.vertline.144143
ribosomal protein S4 [Buchnera aphidicol . . . 255 2.00E-51 HGS036
gi.vertline.2648781 (AE000980) dipeptide ABC transporter, AT . . .
136 1.90E-40 HGS036 gnl.vertline.PID.vertline.e1264523 (AL022121)
putative peptide ABC transpor . . . 185 5.50E-35 HGS036
gi.vertline.143607 sporulation protein [Bacillus subtilis] 191
7.70E-34 HGS036 gnl.vertline.PID.vertline.e1183166 oligopeptide ABC
transporter (ATP-bindin . . . 191 7.70E-34 HGS036
gnl.vertline.PID.vertline.e1253461 oligopeptide transport
ATP-binding prote . . . 213 5.50E-33 HGS036 gi.vertline.2313342
(AE000544) oligopeptide ABC transporter, . . . 258 7.60E-32 HGS036
gnl.vertline.PID.vertline.d1015858 Dipeptide transport ATP-binding
protein . . . 205 1.10E-31 HGS036 gi.vertline.47346 AmiE protein
[Streptococcus pneumoniae] . . . 202 7.40E-31 HGS036
gi.vertline.972897 DppD [Haemophilus influenzae]
>gi.vertline.157411 . . . 204 1.40E-30 HGS036 gi.vertline.677943
AppD [Bacillus subtilis] >gnl.vertline.PID.vertline.e11831 . . .
205 9.70E-30 HGS040 gnl.vertline.PID.vertline.e1185713 elongation
factor P [Bacillus subtilis] . . . 702 7.00E-91 HGS040
gi.vertline.1399829 elongation factor P [Synechococcus PCC79 . . .
541 4.90E-69 HGS040 gnl.vertline.PID.vertline.d1010902 elongation
factor P [Synechocystis sp.] . . . 535 3.20E-68 HGS040
gi.vertline.9581349 ORF1; putative [Anabaena sp.]
>sp.vertline.Q44247 . . . 505 3.80E-64 HGS040
gnl.vertline.PID.vertline.e290977 unknown [Mycobacterium
tuberculosis] >gn . . . 480 9.20E-61 HGS040
gnl.vertline.PID.vertline.e116951- 6 elongation factor P
[Corynebacterium glu . . . 460 4.80E-58 HGS040 gi.vertline.2983772
(AE000736) elongation factor P [Aquifex . . . 435 1.10E-54 HGS040
gi.vertline.1658506 elongation factor P homologue; EF-P [Bac . . .
203 7.20E-52 HGS040 gi.vertline.2313266 (AE000538) translation
elongation factor . . . 409 4.00E-51 HGS040 gi.vertline.536991
elongation factor P [Escherichia coli] >. . . 362 9.40E-45
168153_3 gnl.vertline.PID.vertline.d1028815 (AB009524) Vi
polysaccharide biosynthes . . . 237 5.80E-72 168153_3
gi.vertline.47961 wcdB; ORF3 in citation [1] [Salmonella . . . 234
1.80E-71 168153_3 gi.vertline.1590951 UDP-glucose 4-epimerase
(galE) [Methano . . . 148 3.20E-60 168153_3
pir.vertline.C69149.vertline.C69149 conserved hypothetical protein
MTH380 - . . . 151 1.90E-50 168153_3 gi.vertline.1143204 ORF2;
Method: conceptual translations . . . 227 4.50E-47 168153_3
gnl.vertline.PID.vertline.e316552 unknown [Mycobacterium
tuberculosis] >g . . . 109 4.70E-45 168153_3
gnl.vertline.PID.vertline.e11859- 60 similar to NDP-sugar epimerase
[Bacillu . . . 155 1.80E-39 168153_3
gnl.vertline.PID.vertline.e1289548 (AL023093) putative sugar
dehyratase [M . . . 86 1.80E-36 168153_3 gnl.vertline.PID.vertline-
.e288124 glucose epimerase [Bacillus thuringiensis] 95 2.70E-35
168153_3 gi.vertline.1591707 capsular polysaccharide biosynthesis
pr . . . 85 1.60E-34 168153_2 gnl.vertline.PID.vertline.e1184467
alternate gene name: yvhA [Bacillus subt . . . 354 4.90E-45
168153_2 gi.vertline.1657652 Cap8M [Staphylococcus aureus] 138
9.00E-42 168153_2 gi.vertline.1773352 Cap5M [Staphylococcus aureus]
138 9.00E-42 168153_2 gnl.vertline.PID.vertline.e238668
hypothetical protein [Bacillus subtilis]. . . 139 6.10E-39 168153_2
gi.vertline.1199573 spsB [Sphingomonas sp.] >gi.vertline.1314578
gluc . . . 168 4.40E-35 168153_2 gnl.vertline.PID.vertline.d1005318
ORF14 [Klebsiella pneumoniae] >sp.vertline.Q48460 . . . 260
5.50E-33 168153_2 gnl.vertline.PID.vertline.d1020425 (AB002668)
galactosyltransferase [Actino . . . 155 5.60E-33 168153_2
gnl.vertline.PID.vertline.d1029082 (AB0L0415) glycosyltransferase
[Actinoba . . . 155 2.00E-32 168153_2 gnl.vertline.PID.vertline.d1-
019174 galactosyl-1-phosphate transferase [Syne . . . 139 2.30E-32
168153_2 gnl.vertline.PID.vertline.e220381 structural gene
[Agrobacterium radiobacter] 138 2.40E-32 168153_1
gi.vertline.1276880 EpsG
[Streptococcus thermophilus] 141 3.40E-34 168153_1
gi.vertline.1276879 EpsF [Streptococcus thermophilus] 162 1.70E-29
168153_1 gi.vertline.633699 WbcQ [Yersinia enterocolitica]
>pir.vertline.S512 . . . 134 9.10E-26 168153_1
gnl.vertline.PID.vertline.e238704 hypothetical protein [Bacillus
subtilis]. . . 131 1.90E-18 168153_1 gi.vertline.2983976 (AE000749)
capsular polysaccharide biosy . . . 134 1.50E-15 168153_1
gnl.vertline.PID.vertline.d1005311 ORF7 [Klebsiella pneumoniae]
>sp.vertline.Q484531 . . . 94 2.10E-12 168153_1
gi.vertline.633696 WbcN [Yersinia enterocolitica]
>pir.vertline.S512 . . . 123 2.50E-12 168153_1
gi.vertline.755606 unknown [Bacillus subtilis] 144 5.40E-12
168153_1 gi.vertline.1146237 21.4% of identity to trans-acting
transc . . . 144 6.00E-12 168153_1
gnl.vertline.PID.vertline.e238664 hypothetical protein [Bacillus
subtilis]. . . 141 3.20E-11 168339_2 gnl.vertline.PID.vertline.e11-
69894 putative repeating unit transporter . . . 234 5.70E-57
168339_2 gi.vertline.2209215 (AF004325) putative oligosaccharide .
. . 139 4.90E-37 168339_2 gi.vertline.633692 Wzx [Yersinia
enterocolitica] >pir.vertline.S . . . 141 3.00E-31 168339_2
gi.vertline.2621404 (AE000819) O-antigen transporter [Me . . . 129
8.90E-29 168339_2 gi.vertline.2072448 EpsK [Lactococcus lactis
cremoris] 199 4.00E-27 168339_2 sp.vertline.P37746.vertline.RFBX_E-
COLI PUTATIVE O-ANTIGEN TRANSPORTER. 140 2.10E-23 168339_2
gnl.vertline.PID.vertline.d1016603 Putative O-antigen transporter.
[Esc . . . 140 2.90E-23 168339_2 gi.vertline.510252 membrane
protein [Escherichia coli] 140 8.10E-23 168339_2
gi.vertline.2621427 (AE000822) O-antigen transporter [Me . . . 122
3.10E-20 168339_2 gi.vertline.152778 RFBX [Shigella dysenteriae]
>pir.vertline.S34 . . . 114 8.50E-19
[0090] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only nucleotides outside the 5' and 3'
nucleotides of the subject sequence, as displayed by the FASTDB
alignment, which are not matched/aligned with the query sequence,
are calculated for the purposes of manually adjusting the percent
identity score.
[0091] For example, a 90 nucleotide subject sequence is aligned to
a 100 nucleotide query sequence to determine percent identity. The
deletions occur at the 5' end of the subject sequence and
therefore, the FASTDB alignment does not show a matched/alignment
of the first 10 nucleotides at 5' end. The 10 unpaired nucleotides
represent 10% of the sequence (number of nucleotides at the 5' and
3' ends not matched/total number of nucleotides in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 nucleotides
were perfectly matched the final percent identity would be 90%. In
another example, a 90 nucleotide subject sequence is compared with
a 100 nucleotide query sequence. This time the deletions are
internal deletions so that there are no nucleotides on the 5' or 3'
of the subject sequence which are not matched/aligned with the
query. In this case the percent identity calculated by FASTDB is
not manually corrected. Once again, only nucleotides 5' and 3' of
the subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are made for the purposes of the present invention.
[0092] Vectors and Host Cell
[0093] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
comprising the recombinant vectors, and the production of S. aureus
polypeptides and peptides of the present invention expressed by the
host cells.
[0094] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0095] The S. aureus polynucleotides may be joined to a vector
containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as
a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it may be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0096] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0097] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0098] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0099] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the mature
transcripts expressed by the constructs will preferably include a
translation initiating site at the beginning and a termination
codon (UAA, UGA or UAG) appropriately positioned at the end of the
polypeptide to be translated.
[0100] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin, or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae
or Pichia pastoris (ATCC Accession No. 201178)); insect cells such
as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate
culture mediums and conditions for the above-described host cells
are known in the art.
[0101] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE9, pQE10 available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A available from Stratagene Cloning Systems, Inc.; pET series
of vectors available from Novagen; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in
yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from
Invitrogen, Carlbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan.
[0102] Among known bacterial promoters suitable for use in the
present invention include the E. coli lac I and lacZ promoters, the
T3, T5 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0103] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals (for
example, Davis, et al., Basic Methods In Molecular Biology (1986)).
It is specifically contemplated that the polypeptides of the
present invention may in fact be expressed by a host cell lacking a
recombinant vector.
[0104] Transcription of DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300
nucleotides that act to increase transcriptional activity of a
promoter in a given host cell-type. Examples of enhancers include
the SV40 enhancer, which is located on the late side of the
replication origin at nucleotides 100 to 270, the cytomegalovirus
early promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers.
[0105] For secretion of the translated polypeptide into the lumen
of the endoplasmic reticulum, into the periplasmic space or into
the extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide, for example, the amino
acid sequence KDEL. The signals may be endogenous to the
polypeptide or they may be heterologous signals.
[0106] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL5-receptor has been fused with Fc portions for the purpose
of high-throughput screening assays to identify antagonists of
hIL-5. See Bennett, D. et al. (1995) J. Molec. Recogn. 8:52-58 and
Johanson, K. et al. (1995) J. Biol. Chem. 270 (16):9459-9471.
[0107] The S. aureus polypeptides can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography and high
performance liquid chromatography ("HPLC") is employed for
purification. Polypeptides of the present invention include
naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells.
[0108] Depending upon the host employed in a recombinant production
procedure, the polypeptides of the present invention may be
glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
Thus, it is well known in the art that the N-terminal methionine
encoded by the translation initiation codon generally is removed
with high efficiency from any protein after translation in all
eukaryotic cells. While the N-terminal methionine on most proteins
also is efficiently removed in most prokaryotes, for some proteins,
this prokaryotic removal process is inefficient, depending on the
nature of the amino acid to which the N-terminal methionine is
covalently linked.
[0109] In one embodiment, the yeast Pichia pastoris is used to
express any plasma membrane associated protein of the invention in
a eukaryotic system. Pichia pastoris is a methylotrophic yeast
which can metabolize methanol as its sole carbon source. A main
step in the methanol metabolization pathway is the oxidation of
methanol to formaldehyde using O.sub.2-This reaction is catalyzed
by the enzyme alcohol oxidase. In order to metabolize methanol as
its sole carbon source, Pichia pastoris must generate high levels
of alcohol oxidase due, in part, to the relatively low affinity of
alcohol oxidase for O.sub.2. Consequently, in a growth medium
depending on methanol as a main carbon source, the promoter region
of one of the two alcohol oxidase genes (AOX1) is highly active. In
the presence of methanol, alcohol oxidase produced from the AOX1
gene comprises up to approximately 30% of the total soluble protein
in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol.
5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989);
Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a
heterologous coding sequence, such as, for example, a plasma
membrane associated polynucleotide of the present invention, under
the transcriptional regulation of all or part of the AOX1
regulatory sequence is expressed at exceptionally high-levels in
Pichia yeast grown in the presence of methanol.
[0110] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a plasma membrane associated polypeptide of the
invention, as set forth herein, in a Pichea yeast system
essentially as described in "Pichia Protocols: Methods in Molecular
Biology," D. R. Higgins and J. Cregg, eds. The Humana Press,
Totowa, N.J., 1998. This expression vector allows expression and
secretion of a plasma membrane associated protein of the invention
by virtue of the strong AOX1 promoter linked to the Pichia pastoris
alkaline phosphatase (PHO) secretory signal peptide (i.e., leader)
located upstream of a multiple cloning site.
[0111] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1I/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, and
PAO815, as one skilled in the art would readily appreciate, as long
as the proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0112] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a plasma
membrane associated polynucleotide of the present invention, may be
achieved by cloning the heterologous polynucleotide of the
invention into an expression vector such as, for example, pGAPZ or
pGAPZalpha, and growing the yeast culture in the absence of
methanol.
[0113] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses host
cells that have been engineered to delete or replace endogenous
genetic material (e.g. coding sequences for the polypeptides of the
present invention), and/or to include genetic material (e.g.
heterologous polynucleotide sequences) that is operably associated
with polynucleotides of the present invention, and which activates,
alters, and/or amplifies endogenous polynucleotides of the present
invention. For example, techniques known in the art may be used to
operably associate heterologous control regions (e.g. promoter
and/or enhancer) and endogenous polynucleotide sequences via
homologous recombination (see, e.g. U.S. Pat. No. 5,641,670, issued
Jun. 24, 1997; Internation Publication No. WO 96/29411, published
Sep. 26, 1996; International Publication No. WO 94/12650, published
Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra, et al., Nature 342:435-438
(1989), the disclosures of each of which are hereby incorporated by
reference in their entireties).
[0114] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W. H. Freeman
& Co., N.Y., and Hunkapiller et al., Nature, 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
a polypeptide can be synthesized by use of a peptide synthesizer.
Furthermore, if desired, nonclassical amino acids or chemical amino
acid analogs can be introduced as a substitution or addition into
the polypeptide sequence. Non-classical amino acids include, but
are not limited to, to the D-isomers of the common amino acids,
2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric
acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine,
t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,
fluoro-amino acids, designer amino acids such as b-methyl amino
acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid
analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[0115] Non-naturally occurring variants may be produced using
art-known mutagenesis techniques, which include, but are not
limited to oligonucleotide mediated mutagenesis, alanine scanning,
PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et
al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl.
Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g., Wells
et al., Gene 34:315 (1985)), restriction selection mutagenesis
(see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA
317:415 (1986)).
[0116] The invention additionally, encompasses polypeptides of the
present invention which are differentially modified during or after
translation, such as for example, by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited to: specific chemical cleavage by
cyanogen bromide; trypsin; chymotrypsin; papain; V8 protease;
NaBH.sub.4; acetylation; formylation; oxidation; reduction; and
metabolic synthesis in the presence of tunicamycin, etc.
[0117] Additional post-translational modifications encompassed by
the invention include, for example, N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends,
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0118] Also provided by the invention are chemically modified
derivatives of the polypeptides of the invention which may provide
additional advantages such as increased solubility, stability and
circulating time of the polypeptide, or decreased immunogenicity
(see U.S. Pat. No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0119] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile,
which can include, for example, the duration of sustained release
desired; the effects, if any, on biological activity; the ease in
handling; the degree or lack of antigenicity; and other known
effects of the polyethylene glycol on a therapeutic protein or
analog. For example, the polyethylene glycol may have an average
molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000,
3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,
13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000,
17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000,
35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[0120] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference in their entireties.
[0121] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0122] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to a proteins via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0123] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[0124] As indicated above, pegylation of the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol may be attached to the protein either directly
or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et
al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,
the disclosures of each of which are incorporated herein by
reference.
[0125] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monmethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[0126] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylca- rbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of
which is incorporated herein by reference. Pegylated protein
products produced using the reaction chemistries set out herein are
included within the scope of the invention.
[0127] The number of polyethylene glycol moieties attached to each
protein of the invention (i.e., the degree of substitution) may
also vary. For example, the pegylated proteins of the invention may
be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the
average degree of substitution within ranges such as 1-3,2-4,
3-5,4-6, 5-7,6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15,
14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties
per protein molecule. Methods for determining the degree of
substitution are discussed, for example, in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0128] The polypeptides of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to monomers and
multimers of the polypeptides of the invention, their preparation,
and compositions (preferably, Therapeutics) containing them. In
specific embodiments, the polypeptides of the invention are
monomers, dimers, trimers or tetramers. In additional embodiments,
the multimers of the invention are at least dimers, at least
trimers, or at least tetramers.
[0129] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only polypeptides corresponding to the amino acid
sequence of Table 1 (including fragments, variants, splice
variants, and fusion proteins, corresponding to these as described
herein). These homomers may contain polypeptides having identical
or different amino acid sequences. In a specific embodiment, a
homomer of the invention is a multimer containing only polypeptides
having an identical amino acid sequence. In another specific
embodiment, a homomer of the invention is a multimer containing
polypeptides having different amino acid sequences. In specific
embodiments, the multimer of the invention is a homodimer (e.g.,
containing polypeptides having identical or different amino acid
sequences) or a homotrimer (e.g., containing polypeptides having
identical and/or different amino acid sequences). In additional
embodiments, the homomeric multimer of the invention is at least a
homodimer, at least a homotrimer, or at least a homotetramer.
[0130] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the polypeptides
of the invention. In a specific embodiment, the multimer of the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In
additional embodiments, the heteromeric multimer of the invention
is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
[0131] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the polypeptides of the
invention. Such covalent associations may involve one or more amino
acid residues contained in the polypeptide sequence (e.g., the
polypeptide sequences shown in Table 1). In one instance, the
covalent associations are cross-linking between cysteine residues
located within the polypeptide sequences which interact in the
native (i.e., naturally occurring) polypeptide. In another
instance, the covalent associations are the consequence of chemical
or recombinant manipulation. Alternatively, such covalent
associations may involve one or more amino acid residues contained
in the heterologous polypeptide sequence in a fusion protein.
[0132] In one example, covalent associations are between the
heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in a Fc fusion protein of the invention (as
described herein). In another specific example, covalent
associations of fusion proteins of the invention are between
heterologous polypeptide sequence from another protein that is
capable of forming covalently associated multimers, such as for
example, oseteoprotegerin (see, International Publication NO: WO
98/49305, the contents of which is incorporated herein by reference
in its entirety). In another embodiment, two or more polypeptides
of the invention are joined through peptide linkers. Examples
include those peptide linkers described in U.S. Pat. No. 5,073,627
(incorporated herein by reference in its entirety). Proteins
comprising multiple polypeptides of the invention separated by
peptide linkers may be produced using conventional recombinant DNA
technology.
[0133] Another method for preparing multimer polypeptides of the
invention involves use of polypeptides of the invention fused to a
leucine zipper or isoleucine zipper polypeptide sequence. Leucine
zipper and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been
found in a variety of different proteins. Among the known leucine
zippers are naturally occurring peptides and derivatives thereof
that dimerize or trimerize. Examples of leucine zipper domains
suitable for producing soluble multimeric proteins of the invention
are those described in PCT application WO 94/10308, hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the invention fused to a polypeptide sequence that
dimerizes or trimerizes in solution are expressed in suitable host
cells, and the resulting soluble multimeric fusion protein is
recovered from the culture supernatant using techniques known in
the art.
[0134] Trimeric polypeptides of the invention may offer the
advantage of enhanced biological activity. Preferred leucine zipper
moieties and isoleucine moieties are those that preferentially form
trimers. One example is a leucine zipper derived from lung
surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric polypeptides of the invention.
[0135] In another example, proteins of the invention are associated
by interactions between Flag.RTM. polypeptide sequence contained in
fusion proteins of the invention containing Flag.RTM. polypeptide
seuqence. In a further embodiment, associations proteins of the
invention are associated by interactions between heterologous
polypeptide sequence contained in Flag.RTM. fusion proteins of the
invention and anti-Flag.RTM. antibody.
[0136] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is incorporated herein by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is
incorporated herein by reference in its entirety). Further,
polypeptides of the invention may be routinely modified by the
addition of cysteine or biotin to the C-terminus or N-terminus of
the polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is
incorporated herein by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is incorporated herein by reference in its entirety).
[0137] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is incorporated herein by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is incorporated herein by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hyrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is
incorporated herein by reference in its entirety).
[0138] Polypeptides and Fragments
[0139] The invention further provides an isolated S. aureus
polypeptide having an amino acid sequence in Table 1 or SEQ ID NO:
1 through 61, or a peptide or polypeptide comprising a portion,
fragment, variant or analog of the above polypeptides.
[0140] Variant and Mutant Polypeptides
[0141] To improve or alter the characteristics of S. aureus
polypeptides of the present invention, protein engineering may be
employed. Recombinant DNA technology known to those skilled in the
art can be used to create novel mutant proteins or muteins
including single or multiple amino acid substitutions, deletions,
additions, or fusion proteins. Such modified polypeptides can show,
e.g., increased/decreased activity or increased/decreased
stability. In addition, they may be purified in higher yields and
show better solubility than the corresponding natural polypeptide,
at least under certain purification and storage conditions.
Further, the polypeptides of the present invention may be produced
as multimers including dimers, trimers and tetramers.
Multimerization may be facilitated by linkers or recombinantly
though heterologous polypeptides such as Fc regions.
[0142] N-Terminal and C-Terminal Deletion Mutants
[0143] It is known in the art that one or more amino acids may be
deleted from the N-terminus or C-terminus without substantial loss
of biological function. For instance, Ron et al. J. Biol. Chem.,
268:2984-2988 (1993), reported modified KGF proteins that had
heparin binding activity even if 3, 8, or 27 N-terminal amino acid
residues were missing. Accordingly, the present invention provides
polypeptides having one or more residues deleted from the amino
terminus of the polypeptides shown in Table 1.
[0144] Similarly, many examples of biologically functional
C-terminal deletion mutants are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy-terminus of the protein See,
e.g., Dobeli, et al. (1988) J. Biotechnology 7:199-216.
Accordingly, the present invention provides polypeptides having one
or more residues from the carboxy terminus of the polypeptides
shown in Table 1. The invention also provides polypeptides having
one or more amino acids deleted from both the amino and the
carboxyl termini as described below.
[0145] The present invention is further directed to polynucleotide
encoding portions or fragments of the amino acid sequences
described herein as well as to portions or fragments of the
isolated amino acid sequences described herein. Fragments include
portions of the amino acid sequences of Table 1, at least 7
contiguous amino acid in length, selected from any two integers,
one of which representing a N-terminal position. The first codon of
the polypeptides of Table 1 is position 1. Every combination of a
N-terminal and C-terminal position that a fragment at least 7
contiguous amino acid residues in length could occupy, on any given
amino acid sequence of Table 1 is included in the invention. At
least means a fragment may be 7 contiguous amino acid residues in
length or any integer between 7 and the number of residues in a
full length amino acid sequence minus 1. Therefore, included in the
invention are contiguous fragments specified by any N-terminal and
C-terminal positions of amino acid sequence set forth in Table 1
wherein the contiguous fragment is any integer between 7 and the
number of residues in a full length sequence minus 1.
[0146] Further, the invention includes polypeptides comprising
fragments specified by size, in amino acid residues, rather than by
N-terminal and C-terminal positions. The invention includes any
fragment size, in contiguous amino acid residues, selected from
integers between 7 and the number of residues in a full length
sequence minus 1. Preferred sizes of contiguous polypeptide
fragments include about 7 amino acid residues, about 10 amino acid
residues, about 20 amino acid residues, about 30 amino acid
residues, about 40 amino acid residues, about 50 amino acid
residues, about 100 amino acid residues, about 200 amino acid
residues, about 300 amino acid residues, and about 400 amino acid
residues. The preferred sizes are, of course, meant to exemplify,
not limit, the present invention as all size fragments representing
any integer between 7 and the number of residues in a full length
sequence minus 1 are included in the invention. The present
invention also provides for the exclusion of any fragments
specified by N-terminal and C-terminal positions or by size in
amino acid residues as described above. Any number of fragments
specified by N-terminal and C-terminal positions or by size in
amino acid residues as described above may be excluded.
[0147] The present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the amino
acid sequence of a polypeptide disclosed herein (e.g., any
polypeptide of Table 1). In particular, N-terminal deletions may be
described by the general formula m-q, where q is a whole integer
representing the total number of amino acid residues in a
polypeptide of the invention (e.g., a polypeptide disclosed in
Table 1), and m is defined as any integer ranging from 2 to q-6.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0148] The present invention further provides polypeptides having
one or more residues from the carboxy-terminus of the amino acid
sequence of a polypeptide disclosed herein (e.g., a polypeptide
disclosed in Table 1). In particular, C-terminal deletions may be
described by the general formula 1-n, where n is any whole integer
ranging from 6 to q-1, and where n corresponds to the position of
amino acid residue in a polypeptide of the invention.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0149] In addition, any of the above described N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
polypeptide. The invention also provides polypeptides having one or
more amino acids deleted from both the amino and the carboxyl
termini, which may be described generally as having residues m-n of
a polypeptide encoded by a nucleotide sequence (e.g., including,
but not limited to the preferred polypeptide disclosed in Table 1),
or the cDNA contained in a deposited clone, and/or the complement
thereof, where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0150] The polypeptide fragments of the present invention can be
immediately envisaged using the above description and are therefore
not individually listed solely for the purpose of not unnecessarily
lengthening the specification.
[0151] The above fragments need not be active since they would be
useful, for example, in immunoassays, in epitope mapping, epitope
tagging, to generate antibodies to a particular portion of the
polypeptide, as vaccines, and as molecular weight markers.
[0152] In addition to N- and C-terminal deletion forms of the
protein discussed above, it also will be recognized by one of
ordinary skill in the art that some amino acid sequences of the S.
aureus polypeptides of the present invention can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity.
[0153] Thus, the invention further includes variations of the S.
aureus polypeptides which show substantial S. aureus polypeptide
activity or which include regions of S. aureus protein such as the
protein portions discussed below. Such mutants include deletions,
insertions, inversions, repeats, and substitutions selected
according to general rules known in the art so as to have little
effect on activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided. There
are two main approaches for studying the tolerance of an amino acid
sequence to change. See, Bowie, J. U. et al. (1990), Science
247:1306-1310. The first method relies on the process of evolution,
in which mutations are either accepted or rejected by natural
selection. The second approach uses genetic engineering to
introduce amino acid changes at specific positions of a cloned gene
and selections or screens to identify sequences that maintain
functionality.
[0154] These studies have revealed that proteins are surprisingly
tolerant of amino acid substitutions. The studies indicate which
amino acid changes are likely to be permissive at a certain
position of the protein. For example, most buried amino acid
residues require nonpolar side chains, whereas few features of
surface side chains are generally conserved. Other such
phenotypically silent substitutions are described by Bowie et al.
(supra) and the references cited therein. Typically seen as
conservative substitutions are the replacements, one for another,
among the aliphatic amino acids Ala, Val, Leu and Ile; interchange
of the hydroxyl residues Ser and Thr, exchange of the acidic
residues Asp and Glu, substitution between the amide residues Asn
and Gln, exchange of the basic residues Lys and Arg and
replacements among the aromatic residues Phe, Tyr.
[0155] Thus, the fragment, derivative, analog, or homolog of the
polypeptide of Table 1 may be, for example: (i) one in which one or
more of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code: or (ii) one in which one or more
of the amino acid residues includes a substituent group: or (iii)
one in which the S. aureus polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol): or (iv) one in
which the additional amino acids are fused to the above form of the
polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory sequence or a sequence which is employed for purification
of the above form of the polypeptide or a proprotein sequence. Such
fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0156] Thus, the S. aureus polypeptides of the present invention
may include one or more amino acid substitutions, deletions, or
additions, either from natural mutations or human manipulation. As
indicated, changes are preferably of aminor nature, such as
conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 3).
3TABLE 3 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0157] Amino acids in the S. aureus proteins of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis. See, e.g., Cunningham et al. (1989)
Science 244:1081-1085. The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity using
assays appropriate for measuring the function of the particular
protein.
[0158] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic. See, e.g., Pinckard et al., (1967)
Clin. Exp. Immunol. 2:331-340; Robbins, et al., (1987) Diabetes
36:838-845; Cleland, et al., (1993) Crit. Rev. Therapeutic Drug
Carrier Systems 10:307-377.
[0159] The polypeptides of the present invention are preferably
provided in an isolated form, and may partially or substantially
purified. A recombinantly produced version of the S. aureus
polypeptide can be substantially purified by the one-step method
described by Smith et al. (1988) Gene 67:31-40. Polypeptides of the
invention also can be purified from natural or recombinant sources
using antibodies directed against the polypeptides of the invention
in methods which are well known in the art of protein purification.
The purity of the polypeptide of the present invention may also
specified in percent purity as relative to heterologous containing
polypeptides. Preferred purities include at least 25%, 50%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,
99.75%, and 100% pure, as relative to heretologous containing
polypeptides.
[0160] The invention provides for isolated S. aureus polypeptides
comprising an the amino acid sequence of a full-length S. aureus
polypeptide having the complete amino acid sequence shown in Table
1 and the amino acid sequence of a full-length S. aureus
polypeptide having the complete amino acid sequence shown in Table
1 excepting the N-terminal codon (e.g. including, but not limited
to, methionine, leucine, and/or valine) The polypeptides of the
present invention also include polypeptides having an amino acid
sequence at least 80% identical, more preferably at least 90%
identical, and still more preferably 95%, 96%, 97%, 98% or 99%
identical to a member of the group consisting of (a) a polypeptide
encoded by any of the polynucleotide sequences shown in Table 1,
(b) any of the polypeptide sequences shown in Table 1 and (c) the
complement of a polynucleotide sequence encoding the polypeptide of
(a) or (b) above. Further polypeptides of the present invention
include polypeptides which have at least 90% similarity, more
preferably at least 95% similarity, and still more preferably at
least 96%, 97%, 98% or 99% similarity to those described above.
[0161] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of a S. aureus
polypeptide having an amino acid sequence which contains at least
one conservative amino acid substitution, but not more than 50
conservative amino acid substitutions, not more than 40
conservative amino acid substitutions, not more than 30
conservative amino acid substitutions, and not more than 20
conservative amino acid substitutions. Also provided are
polypeptides which comprise the amino acid sequence of a S. aureus
polypeptide, having at least one, but not more than 10, 9, 8, 7, 6,
5, 4, 3, 2 or 1 conservative amino acid substitutions.
[0162] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% (5 of 100) of the amino acid residues in the
subject sequence may be inserted, deleted, (indels) or substituted
with another amino acid. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0163] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequences shown in Table 1, or a fragment
thereof can be determined conventionally using known computer
programs. A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al., (1990) Comp. App. Biosci.
6:237-245. In a sequence alignment the query and subject sequences
are both amino acid sequences. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=O, Cutoff
Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the subject amino
acid sequence, whichever is shorter.
[0164] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, the results, in percent identity, must be manually
corrected. This is because the FASTDB program does not account for
N- and C-terminal truncations of the subject sequence when
calculating global percent identity. For subject sequences
truncated at the N- and C-termini, relative to the query sequence,
the percent identity is corrected by calculating the number of
residues of the query sequence that are N- and C-terminal of the
subject sequence, which are not matched/aligned with a
corresponding subject residue, as a percent of the total bases of
the query sequence. Whether a residue is matched/aligned is
determined by results of the FASTDB sequence alignment. This
percentage is then subtracted from the percent identity, calculated
by the above FASTDB program using the specified parameters, to
arrive at a final percent identity score. This final percent
identity score is what is used for the purposes of the present
invention. Only residues to the N- and C-termini of the subject
sequence, which are not matched/aligned with the query sequence,
are considered for the purposes of manually adjusting the percent
identity score. That is, only query amino acid residues outside the
farthest N- and C-terminal residues of the subject sequence.
[0165] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not match/align
with the first 10 residues at the N-terminus. The 10 unpaired
residues represent 10% of the sequence (number of residues at the
N- and C-termini not matched/total number of residues in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched the final percent identity would be 90%. In
another example, a 90 residue subject sequence is compared with a
100 residue query sequence. This time the deletions are internal so
there are no residues at the N- or C-termini of the subject
sequence which are not matched/aligned with the query. In this case
the percent identity calculated by FASTDB is not manually
corrected. Once again, only residue positions outside the N- and
C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequence
are manually corrected. No other manual corrections are to made for
the purposes of the present invention.
[0166] The above polypeptide sequences are included irrespective of
whether they have their normal biological activity. This is because
even where a particular polypeptide molecule does not have
biological activity, one of skill in the art would still know how
to use the polypeptide, for instance, as a vaccine or to generate
antibodies. Other uses of the polypeptides of the present invention
that do not have S. aureus activity include, inter alia, as epitope
tags, in epitope mapping, and as molecular weight markers on
SDS-PAGE gels or on molecular sieve gel filtration columns using
methods known to those of skill in the art.
[0167] As described below, the polypeptides of the present
invention can also be used to raise polyclonal and monoclonal
antibodies, which are useful in assays for detecting S. aureus
protein expression or as agonists and antagonists capable of
enhancing or inhibiting S. aureus protein function. Further, such
polypeptides can be used in the yeast two-hybrid system to
"capture" S. aureus protein binding proteins which are also
candidate agonists and antagonists according to the present
invention. See, e.g., Fields et al. (1989) Nature 340:245-246.
[0168] Epitopes and Antibodies
[0169] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence in Table 1, or encoded by a
polynucleotide that hybridizes to the complement of a nucleotide
sequence shown in Table 1 under stringent hybridization conditions
or alternatively, lower stringency hybridization conditions as
defined supra. The present invention further encompasses
polynucleotide sequences encoding an epitope of a polypeptide
sequence of the invention (such as, for example, a nucleotide
sequence disclosed in Table 1), polynucleotide sequences of the
complementary strand of a polynucleotide sequence encoding an
epitope of the invention, and polynucleotide sequences which
hybridize to the complementary strand under stringent hybridization
conditions or lower stringency hybridization conditions defined
supra.
[0170] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0171] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0172] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length. Additional
non-exclusive preferred antigenic epitopes include the antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[0173] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[0174] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0175] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides.
Such fusion proteins may facilitate purification and may increase
half-life in vivo. This has been shown for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of
an antigen across the epithelial barrier to the immune system has
been demonstrated for antigens (e.g., insulin) conjugated to an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG portion
desulfide bonds have also been found to be more efficient in
binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid
in detection and purification of the expressed polypeptide. For
example, a system described by Janknecht et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag
consisting of six histidine residues. The tag serves as a matrix
binding domain for the fusion protein. Extracts from cells infected
with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can
be selectively eluted with imidazole-containing buffers.
[0176] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are incorporated herein by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
those shown in Table 1 and the polypeptides encoded by these
polynucleotides may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments by homologous or
site-specific recombination to generate variation in the
polynucleotide sequence. In another embodiment, polynucleotides of
the invention, or the encoded polypeptides, may be altered by being
subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of a polynucleotide encoding a
polypeptide of the invention may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules.
[0177] Predicted antigenic epitopes are shown in Table 4, below. It
is pointed out that Table 4 only lists amino acid residues
comprising epitopes predicted to have the highest degree of
antigenicity by a particular algorithm. The polypeptides not listed
in Table 4 and portions of polypeptides not listed in Table 4 are
not considered non-antigenic. This is because they may still be
antigenic in vivo but merely not recognized as such by the
particular algorithm used. Thus, Table 4 lists the amino acids
residues comprising only preferred antigenic epitopes, not a
complete list. In fact, all fragments of the polypeptide sequence
of Table 1, at least 7 amino acid residues in length, are included
in the present invention as being useful in epitope mapping and in
making antibodies to particular portions of the polypeptides.
Moreover, Table 4 lists only the critical residues of the epitopes
determined by the Jameson-Wolf analysis. Thus, additional flanking
residues on either the N-terminal, C-terminal, or both N- and
C-terminal ends may be added to the sequences of Table 4 to
generate an epitope-bearing protion a least 7 residues in length.
Amino acid residues comprising other antigenic epitopes may be
determined by algorithms similar to the Jameson-Wolf analysis or by
in vivo testing for an antigenic response using the methods
described herein or those known in the art.
4TABLE 4 Residues Comprising Antigenic Epitoes HGS001 from about
Asp-47 to about Asp-50, from about Ser-128 to about Asp-130, from
about Lys-265 to about Gly-267. HGS005 from about Arg-104 to about
Asp-106, from about Lys-116 to about Lys-120. HGS007m from about
Glu-155 to about Gly-158, from about Gln-178 to about Gly-181, from
about Ser-304 to about Cys-306, from about Asp-401 to about
Tyr-403, from about Asn-405 to about Gly-408, from about Asp-411 to
about Gly-416. HGS009 from about Pro-257 to about Lys-259. HGS014
from about Arg-186 to about Asp-188. HGS019 from about Lys-98 to
about Gly-100, from about Pro-187 to about Asp-189. HGS023 from
about Ser-251 to about Gly-253, from about Lys-437 to about
Lys-440. HGS025 from about Met-51 to about Gly-53. HGS026 from
about Asn-105 to about Lys-108, from about Glu-190 to about
Gly-193, from about Arg-226 to about Ala-230. HGS028 from about
Ile-10 to about Tyr-13. HGS030 from about Glu-11 to about Gly-14,
from about Arg-147 to about Gln-149. HGS033 from about Lys-143 to
about Ser-145. HGS034 from about Pro-33 to about Gln-35. HGS036
from about Asp-64 to about Tyr-66, from about Asp-255 to about
Tyr-257. HGS040 from about Pro-30 to about Lys-32, from about
Asp-76 to about Asp-78. 168153_3 from about Asn-35 to about Arg-37,
from about Pro-135 to about Asp-138, from about Pro-185 to about
Gln-188. 168153_2 from about Asp-54 to about Arg-56. 168153_1 from
about Lys-64 to about Asp-67, from about Gln-319 to about Lys-322,
from about Asn-342 to about Lys-344. 168339_2 from about Asn-82 to
about Arg-85.
[0178] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate an Staphylococcal-specific immune
response or antibodies include fragments of the amino acid
sequences of Table 1 as discussed above. Table 4 discloses a list
of non-limiting residues that are involved in the antigenicity of
the epitope-bearing fragments of the present invention. Therefore,
also included in the present inventions are isolated and purified
antigenic epitope-bearing fragments of the polypeptides of the
present invention comprising a peptide sequences of Table 4. The
antigenic epitope-bearing fragments comprising a peptide sequence
of Table 4 preferably contain between 7 to 50 amino acids (i.e. any
integer between 7 and 50) of a polypeptide of the present
invention. Also, included in the present invention are antigenic
polypeptides between the integers of 7 and the full length sequence
of a polypeptide of Table 1 comprising 1 or more amino acid
sequences of Table 4. Therefore, in most cases, the polypeptides of
Table 4 make up only a portion of the antigenic polypeptide. All
combinations of sequences between the integers of 7 and the full
sequence of a polypeptide sequence of Table 1 are included. The
antigenic epitope-bearing fragments may be specified by either the
number of contiguous amino acid residues or by specific N-terminal
and C-terminal positions as described above for the polypeptide
fragments of the present invention, wherein the first codon of each
polypeptide sequence of Table 1 is position 1. Any number of the
described antigenic epitope-bearing fragments of the present
invention may also be excluded from the present invention in the
same manner.
[0179] Antibodies
[0180] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of a polypeptide
sequence shown in Table 1, and/or an epitope, of the present
invention (as determined by immunoassays well known in the art for
assaying specific antibody-antigen binding). Antibodies of the
invention include, but are not limited to, polyclonal, monoclonal,
multispecific, human, humanized or chimeric antibodies, single
chain antibodies, Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above.
The term "antibody," as used herein, refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that immunospecifically binds an antigen. The immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule.
[0181] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab').sub.2, Fd, single-chain Fvs
(scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments comprising either a VL or VH domain. Antigen-binding
antibody fragments, including single-chain antibodies, may comprise
the variable region(s) alone or in combination with the entirety or
a portion of the following: hinge region, CH1, CH2, and CH3
domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable region(s)
with a hinge region, CH1, CH2, and CH3 domains. The antibodies of
the invention may be from any animal origin including birds and
mammals. Preferably, the antibodies are human, murine (e.g., mouse
and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or
chicken. As used herein, "human" antibodies include antibodies
having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries or
from animals transgenic for one or more human immunoglobulin and
that do not express endogenous immunoglobulins, as described infra
and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et
al.
[0182] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0183] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
sequence listing. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0184] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologs of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10 .sup.7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-1 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5 .times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M.
[0185] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0186] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferrably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0187] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 11
(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9): 1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[0188] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0189] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0190] The antibodies of the invention include derivatives that are
modified, i.e, by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0191] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0192] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0193] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples (e.g., Example 16). In
a non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0194] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0195] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH 1 domain of the heavy chain.
[0196] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0197] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0198] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0199] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0200] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0201] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0202] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0203] Polynucleotides Encoding Antibodies
[0204] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having an amino acid sequence in Table 1.
[0205] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0206] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, for example, a cDNA clone
from a cDNA library that encodes the antibody. Amplified nucleic
acids generated by PCR may then be cloned into replicable cloning
vectors using any method well known in the art.
[0207] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0208] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described above. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0209] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0210] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[0211] Methods of Producing Antibodies
[0212] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0213] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0214] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0215] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0216] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0217] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0218] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0219] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0220] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0221] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0222] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning,
Vol.3. (Academic Press, New York, 1987)). When a marker in the
vector system expressing antibody is amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody gene, production of the antibody
will also increase (Crouse et al., Mol. Cell. Biol. 3:257
(1983)).
[0223] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0224] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0225] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452 (1991), which are incorporated by
reference in their entireties.
[0226] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341(1992) (said references incorporated by
reference in their entireties).
[0227] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of a polypeptide
sequence shown in Table 1 may be fused or conjugated to the above
antibody portions to increase the in vivo half life of the
polypeptides or for use in immunoassays using methods known in the
art. Further, any one of the polypeptides shown in Table 1 may be
fused or conjugated to the above antibody portions to facilitate
purification. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0228] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0229] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or 99Tc.
[0230] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0231] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0232] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0233] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0234] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0235] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[0236] Immunophenotyping
[0237] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0238] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
[0239] Assays For Antibody Binding
[0240] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0241] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0242] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0243] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0244] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
[0245] Therapeutic Uses
[0246] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein. The treatment and/or prevention of
diseases, disorders, or conditions associated with aberrant
expression and/or activity of a polypeptide of the invention
includes, but is not limited to, alleviating symptoms associated
with those diseases, disorders or conditions. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0247] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0248] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0249] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0250] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 5.times.10.sup.-14 M, 5.times.10.sup.-14 M,
5.times.10.sup.-15 M, and 10.sup.-15 M.
[0251] Gene Therapy
[0252] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0253] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0254] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0255] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both the heavy and light chains, or fragments thereof, of
the antibody.
[0256] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0257] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0258] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[0259] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0260] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0261] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0262] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0263] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0264] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0265] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0266] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0267] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription. Demonstration of
Therapeutic or Prophylactic Activity
[0268] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[0269] Therapeutic/Prophylactic Administration and Composition
[0270] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0271] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0272] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0273] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0274] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0275] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0276] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0277] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox--like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0278] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0279] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0280] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0281] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0282] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0283] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. Diagnosis and Imaging
[0284] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or disorders associated with the aberrant expression and/or
activity of a polypeptide of the invention. The invention provides
for the detection of aberrant expression of a polypeptide of
interest, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0285] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0286] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0287] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0288] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99 mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[0289] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0290] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0291] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0292] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
[0293] Kits
[0294] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0295] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0296] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0297] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0298] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0299] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0300] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0301] Diagnostic Assays
[0302] The present invention further relates to methods for
assaying staphylococcal infection in an animal (e.g., a mammal,
including but not limited to a human) by detecting the expression
of genes encoding staphylococcal polypeptides of the present
invention. The methods comprise analyzing tissue or body fluid from
the animal for Staphylococcus-specific antibodies, nucleic acids,
or proteins. Analysis of nucleic acid specific to Staphylococcus is
assayed by PCR or hybridization techniques using nucleic acid
sequences of the present invention as either hybridization probes
or primers. See, e.g., Sambrook et al. Molecular cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.,
1989, page 54 reference); Eremeeva et al. (1994) J. Clin.
Microbiol. 32:803-810 (describing differentiation among spotted
fever group Rickettsiae species by analysis of restriction fragment
length polymorphism of PCR-amplified DNA) and Chen et al. 1994 J.
Clin. Microbiol. 32:589-595 (detecting bacterial nucleic acids via
PCR).
[0303] Where diagnosis of a disease state related to infection with
Staphylococcus has already been made, the present invention is
useful for monitoring progression or regression of the disease
state by measuring the amount of Staphylococcus cells present in a
patient or whereby patients exhibiting enhanced Staphylococcus gene
expression will experience a worse clinical outcome relative to
patients expressing these gene(s) at a lower level.
[0304] By "biological sample" is intended any biological sample
obtained from an animal, cell line, tissue culture, or other source
which contains Staphylococcus polypeptide, mRNA, or DNA. Biological
samples include body fluids (such as saliva, blood, plasma, urine,
mucus, synovial fluid, etc.) tissues (such as muscle, skin, and
cartilage) and any other biological source suspected of containing
Staphylococcus polypeptides or nucleic acids. Methods for obtaining
biological samples such as tissue are well known in the art.
[0305] The present invention is useful for detecting diseases
related to Staphylococcus infections in animals. Preferred animals
include monkeys, apes, cats, dogs, birds, cows, pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
[0306] Total RNA can be isolated from a biological sample using any
suitable technique such as the single-step
guanidinium-thiocyanate-phenol- -chloroform method described in
Chomczynski et al. (1987) Anal. Biochem. 162:156-159. mRNA encoding
Staphylococcus polypeptides having sufficient homology to the
nucleic acid sequences identified in Table 1 to allow for
hybridization between complementary sequences are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0307] Northern blot analysis can be performed as described in
Harada et al. (1990) Cell 63:303-312. Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. A S. aureus polynucleotide
sequence shown in Table 1 labeled according to any appropriate
method (such as the .sup.32P-multiprimed DNA labeling system
(Amersham)) is used as probe. After hybridization overnight, the
filter is washed and exposed to x-ray film. DNA for use as probe
according to the present invention is described in the sections
above and will preferably at least 15 nucleotides in length.
[0308] S1 mapping can be performed as described in Fujita et al.
(1987) Cell 49:357-367. To prepare probe DNA for use in S1 mapping,
the sense strand of an above-described S. aureus DNA sequence of
the present invention is used as a template to synthesize labeled
antisense DNA. The antisense DNA can then be digested using an
appropriate restriction endonuclease to generate further DNA probes
of a desired length. Such antisense probes are useful for
visualizing protected bands corresponding to the target mRNA (i.e.,
mRNA encoding polypeptides of the present invention).
[0309] Levels of mRNA encoding Staphylococcus polypeptides are
assayed, for e.g., using the RT-PCR method described in Makino et
al. (1990) Technique 2:295-301. By this method, the radioactivities
of the "amplicons" in the polyacrylamide gel bands are linearly
related to the initial concentration of the target mRNA. Briefly,
this method involves adding total RNA isolated from a biological
sample in a reaction mixture containing a RT primer and appropriate
buffer. After incubating for primer annealing, the mixture can be
supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and
reverse transcriptase. After incubation to achieve reverse
transcription of the RNA, the RT products are then subject to PCR
using labeled primers. Alternatively, rather than labeling the
primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding the Staphylococcus polypeptides of the present invention)
are quantified using an imaging analyzer. RT and PCR reaction
ingredients and conditions, reagent and gel concentrations, and
labeling methods are well known in the art. Variations on the
RT-PCR method will be apparent to the skilled artisan. Other PCR
methods that can detect the nucleic acid of the present invention
can be found in PCR PRIMER: A LABORATORY MANUAL (C. W. Dieffenbach
et al. eds., Cold Spring Harbor Lab Press, 1995).
[0310] The polynucleotides of the present invention, including both
DNA and RNA, may be used to detect polynucleotides of the present
invention or Staphylococcus species including S. aureus using bio
chip technology. The present invention includes both high density
chip arrays (>1000 oligonucleotides per cm.sup.2) and low
density chip arrays (<1000 oligonucleotides per cm.sup.2). Bio
chips comprising arrays of polynucleotides of the present invention
may be used to detect Staphylococcus species, including S. aureus,
in biological and environmental samples and to diagnose an animal,
including humans, with an S. aureus or other Staphylococcus
infection. The bio chips of the present invention may comprise
polynucleotide sequences of other pathogens including bacteria,
viral, parasitic, and fungal polynucleotide sequences, in addition
to the polynucleotide sequences of the present invention, for use
in rapid differential pathogenic detection and diagnosis. The bio
chips can also be used to monitor an S. aureus or other
Staphylococcus infections and to monitor the genetic changes
(deletions, insertions, mismatches, etc.) in response to drug
therapy in the clinic and drug development in the laboratory. The
bio chip technology comprising arrays of polynucleotides of the
present invention may also be used to simultaneously monitor the
expression of a multiplicity of genes, including those of the
present invention. The polynucleotides used to comprise a selected
array may be specified in the same manner as for the fragments,
i.e, by their 5' and 3' positions or length in contigious base
pairs and include from. Methods and particular uses of the
polynucleotides of the present invention to detect Staphylococcus
species, including S. aureus, using bio chip technology include
those known in the art and those of: U.S. Pat. Nos. 5,510,270,
5,545,531, 5445934, 5677195, 5532128, 5556752, 5527681, 5451683,
5424186, 5607646, 5658732 and World Patent Nos. WO/9710365,
WO/9511995, WO/9743447, WO/9535505, each incorporated herein in
their entireties.
[0311] Biosensors using the polynucleotides of the present
invention may also be used to detect, diagnose, and monitor S.
aureus or other Staphylococcus species and infections thereof.
Biosensors using the polynucleotides of the present invention may
also be used to detect particular polynucleotides of the present
invention. Biosensors using the polynucleotides of the present
invention may also be used to monitor the genetic changes
(deletions, insertions, mismatches, etc.) in response to drug
therapy in the clinic and drug development in the laboratory.
Methods and particular uses of the polynucleotides of the present
invention to detect Staphylococcus species, including S. aureus,
using biosenors include those known in the art and those of: U.S.
Pat. Nos. 5,721,102, 5,658,732, 5631170, and World Patent Nos.
WO97/35011, WO/9720203, each incorporated herein in their
entireties.
[0312] Thus, the present invention includes both bio chips and
biosensors comprising polynucleotides of the present invention and
methods of their use.
[0313] A preferred composition of matter comprises isolated nucleic
acid molecules wherein the nucleotide sequences of said nucleic
acid molecules comprise a bio chip or biosensor of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200,
250, 300, 500, 1000, 2000, 3000 or 4000 nucleotide sequences,
wherein at least one sequence in said DNA bio chip or biosensor is
at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a S. aureus polynucleotide shown in Table 1. The
nucleic acid molecules can comprise DNA molecules or RNA
molecules.
[0314] Assaying Staphylococcus polypeptide levels in a biological
sample can occur using any art-known method, such as antibody-based
techniques. For example, Staphylococcus polypeptide expression in
tissues can be studied with classical immunohistological methods.
In these, the specific recognition is provided by the primary
antibody (polyclonal or monoclonal) but the secondary detection
system can utilize fluorescent, enzyme, or other conjugated
secondary antibodies. As a result, an immunohistological staining
of tissue section for pathological examination is obtained. Tissues
can also be extracted, e.g., with urea and neutral detergent, for
the liberation of Staphylococcus polypeptides for Western-blot or
dot/slot assay. See, e.g., Jalkanen, M. et al. (1985) J. Cell.
Biol. 101:976-985; Jalkanen, M. et al. (1987) J. Cell. Biol.
105:3087-3096. In this technique, which is based on the use of
cationic solid phases, quantitation of a Staphylococcus polypeptide
can be accomplished using an isolated Staphylococcus polypeptide as
a standard. This technique can also be applied to body fluids.
[0315] Other antibody-based methods useful for detecting
Staphylococcus polypeptide gene expression include immunoassays,
such as the ELISA and the radioimmunoassay (RIA). For example, a
Staphylococcus polypeptide-specific monoclonal antibodies can be
used both as an immunoabsorbent and as an enzyme-labeled probe to
detect and quantify a Staphylococcus polypeptide. The amount of a
Staphylococcus polypeptide present in the sample can be calculated
by reference to the amount present in a standard preparation using
a linear regression computer algorithm. Such an ELISA is described
in Iacobelli et al. (1988) Breast Cancer Research and Treatment
11:19-30. In another ELISA assay, two distinct specific monoclonal
antibodies can be used to detect Staphylococcus polypeptides in a
body fluid. In this assay, one of the antibodies is used as the
immunoabsorbent and the other as the enzyme-labeled probe.
[0316] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting the Staphylococcus polypeptide with immobilized antibody
and, without washing, contacting the mixture with the labeled
antibody. The "two-step" assay involves washing before contacting
the mixture with the labeled antibody. Other conventional methods
may also be employed as suitable. It is usually desirable to
immobilize one component of the assay system on a support, thereby
allowing other components of the system to be brought into contact
with the component and readily removed from the sample. Variations
of the above and other immunological methods included in the
present invention can also be found in Harlow et al., ANTIBODIES: A
LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988).
[0317] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labeled antibody/substrate reaction. Besides enzymes, other
suitable labels include radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulphur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0318] Further suitable labels for the Staphylococcus
polypeptide-specific antibodies of the present invention are
provided below. Examples of suitable enzyme labels include malate
dehydrogenase, Staphylococcus nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase.
[0319] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32p, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Su, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212 Pb,
.sup.47SC, .sup.109Pd, etc. .sup.111In is a preferred isotope where
in vivo imaging is used since its avoids the problem of
dehalogenation of the .sup.125I or .sup.131I-labeled monoclonal
antibody by the liver. In addition, this radionucleotide has a more
favorable gamma emission energy for imaging. See, e.g., Perkins et
al. (1985) Eur. J. Nucl. Med. 10:296-301; Carasquillo et al. (1987)
J. Nucl. Med. 28:281-287. For example, .sup.11 In coupled to
monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA has
shown little uptake in non-tumors tissues, particularly the liver,
and therefore enhances specificity of tumor localization. See,
Esteban et al. (1987) J. Nucl. Med. 28:861-870.
[0320] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, 55NM, .sup.162Dy, 12Tr, and 56Fe.
[0321] Examples of suitable fluorescent labels include an 152Eu
label, a fluorescein label, an isothiocyanate label, a rhodamine
label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0322] Examples of suitable toxin labels include, Pseudomonas
toxin, diphtheria toxin, ricin, and cholera toxin.
[0323] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0324] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and iron.
[0325] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (1976) Clin. Chim. Acta
70:1-31, and Schurs et al. (1977) Clin. Chim. Acta 81:1-40.
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy- -succinimide ester method, all of
which methods are incorporated by reference herein.
[0326] In a related aspect, the invention includes a diagnostic kit
for use in screening serum containing antibodies specific against
S. aureus infection. Such a kit may include an isolated S. aureus
antigen comprising an epitope which is specifically immunoreactive
with at least one anti-S. aureus antibody. Such a kit also includes
means for detecting the binding of said antibody to the antigen. In
specific embodiments, the kit may include a recombinantly produced
or chemically synthesized peptide or polypeptide antigen. The
peptide or polypeptide antigen may be attached to a solid
support.
[0327] In a more specific embodiment, the detecting means of the
above-described kit includes a solid support to which said peptide
or polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the S. aureus antigen can be
detected by binding of the reporter labeled antibody to the anti-S.
aureus polypeptide antibody.
[0328] In a related aspect, the invention includes a method of
detecting S. aureus infection in a subject. This detection method
includes reacting a body fluid, preferably serum, from the subject
with an isolated S. aureus antigen, and examining the antigen for
the presence of bound antibody. In a specific embodiment, the
method includes a polypeptide antigen attached to a solid support,
and serum is reacted with the support. Subsequently, the support is
reacted with a reporter-labeled anti-human antibody. The support is
then examined for the presence of reporter-labeled antibody.
[0329] The solid surface reagent employed in the above assays and
kits is prepared by known techniques for attaching protein material
to solid support material, such as polymeric beads, dip sticks,
96-well plates or filter material. These attachment methods
generally include non-specific adsorption of the protein to the
support or covalent attachment of the protein, typically through a
free amine group, to a chemically reactive group on the solid
support, such as an activated carboxyl, hydroxyl, or aldehyde
group. Alternatively, streptavidin coated plates can be used in
conjunction with biotinylated antigen(s).
[0330] The polypeptides and antibodies of the present invention,
including fragments thereof, may be used to detect Staphylococcus
species including S. aureus using bio chip and biosensor
technology. Bio chip and biosensors of the present invention may
comprise the polypeptides of the present invention to detect
antibodies, which specifically recognize Staphylococcus species,
including S. aureus. Bio chip and biosensors of the present
invention may also comprise antibodies which specifically recognize
the polypeptides of the present invention to detect Staphylococcus
species, including S. aureus or specific polypeptides of the
present invention. Bio chips or biosensors comprising polypeptides
or antibodies of the present invention may be used to detect
Staphylococcus species, including S. aureus, in biological and
environmental samples and to diagnose an animal, including humans,
with an S. aureus or other Staphylococcus infection. Thus, the
present invention includes both bio chips and biosensors comprising
polypeptides or antibodies of the present invention and methods of
their use.
[0331] The bio chips of the present invention may further comprise
polypeptide sequences of other pathogens including bacteria, viral,
parasitic, and fungal polypeptide sequences, in addition to the
polypeptide sequences of the present invention, for use in rapid
diffenertial pathogenic detection and diagnosis. The bio chips of
the present invention may further comprise antibodies or fragements
thereof specific for other pathogens including bacteria, viral,
parasitic, and fungal polypeptide sequences, in addition to the
antibodies or fragements thereof of the present invention, for use
in rapid diffenertial pathogenic detection and diagnosis. The bio
chips and biosensors of the present invention may also be used to
monitor an S. aureus or other Staphylococcus infection and to
monitor the genetic changes (amio acid deletions, insertions,
substitutions, etc.) in response to drug therapy in the clinic and
drug development in the laboratory. The bio chip and biosensors
comprising polypeptides or antibodies of the present invention may
also be used to simultaneously monitor the expression of a
multiplicity of polypeptides, including those of the present
invention. The polypeptides used to comprise a bio chip or
biosensor of the present invention may be specified in the same
manner as for the fragements, i.e, by their N-terminal and
C-terminal positions or length in contigious amino acid residue.
Methods and particular uses of the polypeptides and antibodies of
the present invention to detect Staphylococcus species, including
S. aureus, or specific polypeptides using bio chip and biosensor
technology include those known in the art, those of the U.S. Patent
Nos. and World Patent Nos. listed above for bio chips and
biosensors using polynucleotides of the present invention, and
those of: U.S. Pat. Nos. 5,658,732, 5,135,852, 5567301, 5677196,
5690894 and World Patent Nos. WO9729366, WO9612957, each
incorporated herein in their entireties.
[0332] Treatment
[0333] Agonists and Antagonists--Assays and Molecules
[0334] The invention also provides a method of screening compounds
to identify those which enhance or block the biological activity of
the S. aureus polypeptides of the present invention. The present
invention further provides where the compounds kill or slow the
growth of S. aureus. The ability of S. aureus antagonists,
including S. aureus ligands, to prophylactically or therapeutically
block antibiotic resistance may be easily tested by the skilled
artisan. See, e.g., Straden et al. (1997) J. Bacteriol.
179(1):9-16.
[0335] An agonist is a compound which increases the natural
biological function or which functions in a manner similar to the
polypeptides of the present invention, while antagonists decrease
or eliminate such functions. Potential antagonists include small
organic molecules, peptides, polypeptides, and antibodies that bind
to a polypeptide of the invention and thereby inhibit or extinguish
its activity.
[0336] The antagonists may be employed for instance to inhibit
peptidoglycan cross bridge formation. Antibodies against S. aureus
may be employed to bind to and inhibit S. aureus activity to treat
antibiotic resistance. Any of the above antagonists may be employed
in a composition with a pharmaceutically acceptable carrier.
[0337] Vaccines
[0338] The present invention also provides vaccines comprising one
or more polypeptides of the present invention. Heterogeneity in the
composition of a vaccine may be provided by combining S. aureus
polypeptides of the present invention. Multi-component vaccines of
this type are desirable because they are likely to be more
effective in eliciting protective immune responses against multiple
species and strains of the Staphylococcus genus than single
polypeptide vaccines.
[0339] Multi-component vaccines are known in the art to elicit
antibody production to numerous immunogenic components. See, e.g.,
Decker et al. (1996) J. Infect. Dis. 174:S270-275. In addition, a
hepatitis B, diphtheria, tetanus, pertussis tetravalent vaccine has
recently been demonstrated to elicit protective levels of
antibodies in human infants against all four pathogenic agents.
See, e.g., Aristegui, J. et al. (1997) Vaccine 15:7-9.
[0340] The present invention in addition to single-component
vaccines includes multi-component vaccines. These vaccines comprise
more than one polypeptide, immunogen or antigen. Thus, a
multi-component vaccine would be a vaccine comprising more than one
of the S. aureus polypeptides of the present invention.
[0341] Further within the scope of the invention are whole cell and
whole viral vaccines. Such vaccines may be produced recombinantly
and involve the expression of one or more of the S. aureus
polypeptides described in Table 1. For example, the S. aureus
polypeptides of the present invention may be either secreted or
localized intracellular, on the cell surface, or in the periplasmic
space. Further, when a recombinant virus is used, the S. aureus
polypeptides of the present invention may, for example, be
localized in the viral envelope, on the surface of the capsid, or
internally within the capsid. Whole cells vaccines which employ
cells expressing heterologous proteins are known in the art. See,
e.g., Robinson, K. et al. (1997) Nature Biotech. 15:653-657;
Sirard, J. et al. (1997) Infect. Immun. 65:2029-2033; Chabalgoity,
J. et al. (1997) Infect. Immun. 65:2402-2412. These cells may be
administered live or may be killed prior to administration.
Chabalgoity, J. et al., supra, for example, report the successful
use in mice of a live attenuated Salmonella vaccine strain which
expresses a portion of a platyhelminth fatty acid-binding protein
as a fusion protein on its cells surface.
[0342] A multi-component vaccine can also be prepared using
techniques known in the art by combining one or more S. aureus
polypeptides of the present invention, or fragments thereof, with
additional non-staphylococcal components (e.g., diphtheria toxin or
tetanus toxin, and/or other compounds known to elicit an immune
response). Such vaccines are useful for eliciting protective immune
responses to both members of the Staphylococcus genus and
non-staphylococcal pathogenic agents.
[0343] The vaccines of the present invention also include DNA
vaccines. DNA vaccines are currently being developed for a number
of infectious diseases. See, et al., Boyer, et al. (1997) Nat. Med.
3:526-532; reviewed in Spier, R. (1996) Vaccine 14:1285-1288. Such
DNA vaccines contain a nucleotide sequence encoding one or more S.
aureus polypeptides of the present invention oriented in a manner
that allows for expression of the subject polypeptide. For example,
the direct administration of plasmid DNA encoding B. burgdorgeri
OspA has been shown to elicit protective immunity in mice against
borrelial challenge. See, Luke et al. (1997) J. Infect. Dis.
175:91-97.
[0344] The present invention also relates to the administration of
a vaccine which is co-administered with a molecule capable of
modulating immune responses. Kim et al. (1997) Nature Biotech.
15:641-646, for example, report the enhancement of immune responses
produced by DNA immunizations when DNA sequences encoding molecules
which stimulate the immune response are co-administered. In a
similar fashion, the vaccines of the present invention may be
co-administered with either nucleic acids encoding immune
modulators or the immune modulators themselves. These immune
modulators include granulocyte macrophage colony stimulating factor
(GM-CSF) and CD86.
[0345] The vaccines of the present invention may be used to confer
resistance to staphylococcal infection by either passive or active
immunization. When the vaccines of the present invention are used
to confer resistance to staphylococcal infection through active
immunization, a vaccine of the present invention is administered to
an animal to elicit a protective immune response which either
prevents or attenuates a staphylococcal infection. When the
vaccines of the present invention are used to confer resistance to
staphylococcal infection through passive immunization, the vaccine
is provided to a host animal (e.g., human, dog, or mouse), and the
antisera elicited by this antisera is recovered and directly
provided to a recipient suspected of having an infection caused by
a member of the Staphylococcus genus.
[0346] The ability to label antibodies, or fragments of antibodies,
with toxin molecules provides an additional method for treating
staphylococcal infections when passive immunization is conducted.
In this embodiment, antibodies, or fragments of antibodies, capable
of recognizing the S. aureus polypeptides disclosed herein, or
fragments thereof, as well as other Staphylococcus proteins, are
labeled with toxin molecules prior to their administration to the
patient. When such toxin derivatized antibodies bind to
Staphylococcus cells, toxin moieties will be localized to these
cells and will cause their death.
[0347] The present invention thus concerns and provides a means for
preventing or attenuating a staphylococcal infection resulting from
organisms which have antigens that are recognized and bound by
antisera produced in response to the polypeptides of the present
invention. As used herein, a vaccine is said to prevent or
attenuate a disease if its administration to an animal results
either in the total or partial attenuation (i.e., suppression) of a
symptom or condition of the disease, or in the total or partial
immunity of the animal to the disease.
[0348] The administration of the vaccine (or the antisera which it
elicits) may be for either a "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the compound(s) are
provided in advance of any symptoms of staphylococcal infection.
The prophylactic administration of the compound(s) serves to
prevent or attenuate any subsequent infection. When provided
therapeutically, the compound(s) is provided upon or after the
detection of symptoms which indicate that an animal may be infected
with a member of the Staphylococcus genus. The therapeutic
administration of the compound(s) serves to attenuate any actual
infection. Thus, the S. aureus polypeptides, and fragments thereof,
of the present invention may be provided either prior to the onset
of infection (so as to prevent or attenuate an anticipated
infection) or after the initiation of an actual infection.
[0349] The polypeptides of the invention, whether encoding a
portion of a native protein or a functional derivative thereof, may
be administered in pure form or may be coupled to a macromolecular
carrier. Example of such carriers are proteins and carbohydrates.
Suitable proteins which may act as macromolecular carrier for
enhancing the immunogenicity of the polypeptides of the present
invention include keyhole limpet hemacyanin (KLH) tetanus toxoid,
pertussis toxin, bovine serum albumin, and ovalbumin. Methods for
coupling the polypeptides of the present invention to such
macromolecular carriers are disclosed in Harlow et al., ANTIBODIES:
A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988).
[0350] A composition is said to be "pharmacologically or
physiologically acceptable" if its administration can be tolerated
by a recipient animal and is otherwise suitable for administration
to that animal. Such an agent is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient.
[0351] While in all instances the vaccine of the present invention
is administered as a pharmacologically acceptable compound, one
skilled in the art would recognize that the composition of a
pharmacologically acceptable compound varies with the animal to
which it is administered. For example, a vaccine intended for human
use will generally not be co-administered with Freund's adjuvant.
Further, the level of purity of the S. aureus polypeptides of the
present invention will normally be higher when administered to a
human than when administered to a non-human animal.
[0352] As would be understood by one of ordinary skill in the art,
when the vaccine of the present invention is provided to an animal,
it may be in a composition which may contain salts, buffers,
adjuvants, or other substances which are desirable for improving
the efficacy of the composition. Adjuvants are substances that can
be used to specifically augment a specific immune response. These
substances generally perform two functions: (1) they protect the
antigen(s) from being rapidly catabolized after administration and
(2) they nonspecifically stimulate immune responses.
[0353] Normally, the adjuvant and the composition are mixed prior
to presentation to the immune system, or presented separately, but
into the same site of the animal being immunized. Adjuvants can be
loosely divided into several groups based upon their composition.
These groups include oil adjuvants (for example, Freund's complete
and incomplete), mineral salts (for example, AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH4(SO.sub.4), silica, kaolin, and carbon),
polynucleotides (for example, poly IC and poly AU acids), and
certain natural substances (for example, wax D from Mycobacterium
tuberculosis, as well as substances found in Corynebacterium
parvum, or Bordetella pertussis, and members of the genus Brucella.
Other substances useful as adjuvants are the saponins such as, for
example, Quil A. (Superfos A/S, Denmark). Preferred adjuvants for
use in the present invention include aluminum salts, such as
AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2, and AlNH4(SO.sub.4).
Examples of materials suitable for use in vaccine compositions are
provided in REMINGTON'S PHARMACEUTICAL SCIENCES 1324-1341 (A. Osol,
ed, Mack Publishing Co, Easton, Pa., (1980) (incorporated herein by
reference).
[0354] The therapeutic compositions of the present invention can be
administered parenterally by injection, rapid infusion,
nasopharyngeal absorption (intranasopharangeally), dermoabsorption,
or orally. The compositions may alternatively be administered
intramuscularly, or intravenously. Compositions for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Carriers
or occlusive dressings can be used to increase skin permeability
and enhance antigen absorption. Liquid dosage forms for oral
administration may generally comprise a liposome solution
containing the liquid dosage form. Suitable forms for suspending
liposomes include emulsions, suspensions, solutions, syrups, and
elixirs containing inert diluents commonly used in the art, such as
purified water. Besides the inert diluents, such compositions can
also include adjuvants, wetting agents, emulsifying and suspending
agents, or sweetening, flavoring, or perfuming agents.
[0355] Therapeutic compositions of the present invention can also
be administered in encapsulated form. For example, intranasal
immunization using vaccines encapsulated in biodegradable
microsphere composed of poly(DL-lactide-co-glycolide). See, Shahin,
R. et al. (1995) Infect. Immun. 63:1195-1200. Similarly, orally
administered encapsulated Salmonella typhimurium antigens can also
be used. Allaoui-Attarki, K. et al. (1997) Infect. Immun.
65:853-857. Encapsulated vaccines of the present invention can be
administered by a variety of routes including those involving
contacting the vaccine with mucous membranes (e.g., intranasally,
intracolonicly, intraduodenally).
[0356] Many different techniques exist for the timing of the
immunizations when a multiple administration regimen is utilized.
It is possible to use the compositions of the invention more than
once to increase the levels and diversities of expression of the
immunoglobulin repertoire expressed by the immunized animal.
Typically, if multiple immunizations are given, they will be given
one to two months apart.
[0357] According to the present invention, an "effective amount" of
a therapeutic composition is one which is sufficient to achieve a
desired biological effect. Generally, the dosage needed to provide
an effective amount of the composition will vary depending upon
such factors as the animal's or human's age, condition, sex, and
extent of disease, if any, and other variables which can be
adjusted by one of ordinary skill in the art.
[0358] The antigenic preparations of the invention can be
administered by either single or multiple dosages of an effective
amount. Effective amounts of the compositions of the invention can
vary from 0.01-1,000 .mu.g/ml per dose, more preferably 0.1-500
.mu.g/ml per dose, and most preferably 10-300 .mu.g/ml per
dose.
EXAMPLES
Example 1
Isolation of a Selected DNA Clone From the Deposited Sample
[0359] Three approaches can be used to isolate a S. aureus clone
comprising a polynucleotide of the present invention from any S.
aureus genomic DNA library. The S. aureus strain ISP3 has been
deposited as a convienent source for obtaining a S. aureus strain
although a wide varity of strains S. aureus strains can be used
which are known in the art.
[0360] S. aureus genomic DNA is prepared using the following
method. A 20 ml overnight bacterial culture grown in a rich medium
(e.g., Trypticase Soy Broth, Brain Heart Infusion broth or Super
broth), pelleted, washed two times with TES (30 mM Tris-pH 8.0, 25
mM EDTA, 50 mM NaCl), and resuspended in 5 ml high salt TES (2.5M
NaCl). Lysostaphin is added to final concentration of approx 50
.mu.g/ml and the mixture is rotated slowly 1 hour at 37C to make
protoplast cells. The solution is then placed in incubator (or
place in a shaking water bath) and warmed to 55C. Five hundred
micro liter of 20% sarcosyl in TES (final concentration 2%) is then
added to lyse the cells. Next, guanidine HCl is added to a final
concentration of 7M (3.69g in 5.5 ml). The mixture is swirled
slowly at 55C for 60-90 min (solution should clear). A CsCl
gradient is then set up in SW41 ultra clear tubes using 2.0 ml 5.7M
CsCl and overlaying with 2.85M CsCl. The gradient is carefully
overlayed with the DNA-containing GuHCl solution. The gradient is
spun at 30,000 rpm, 20C for 24 hr and the lower DNA band is
collected. The volume is increased to 5 ml with TE buffer. The DNA
is then treated with protease K (10 .mu.g/1 ml) overnight at 37 C,
and precipitated with ethanol. The precipitated DNA is resuspended
in a desired buffer.
[0361] In the first method, a plasmid is directly isolated by
screening a plasmid S. aureus genomic DNA library using a
polynucleotide probe corresponding to a polynucleotide of the
present invention. Particularly, a specific polynucleotide with
30-40 nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
is labeled, for instance, with .sup.32P-.gamma.-ATP using T4
polynucleotide kinase and purified according to routine methods.
(See, e.g., Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The
library is transformed into a suitable host, as indicated above
(such as XL-1 Blue (Stratagene)) using techniques known to those of
skill in the art. See, e.g., Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel
et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley and
Sons, N.Y. 1989). The transformants are plated on 1.5% agar plates
(containing the appropriate selection agent, e.g., ampicillin) to a
density of about 150 transformants (colonies) per plate. These
plates are screened using Nylon membranes according to routine
methods for bacterial colony screening. See, e.g., Sambrook et al.
MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor, N.Y.
2nd ed. 1989); Ausubel et al., CURRENT PROTOCALS IN MOLECULAR
BIOLOGY (John Wiley and Sons, N.Y. 1989) or other techniques known
to those of skill in the art.
[0362] Alternatively, two primers of 15-25 nucleotides derived from
the 5' and 3' ends of a polynucleotide of Table 1 are synthesized
and used to amplify the desired DNA by PCR using a S. aureus
genomic DNA prep (e.g., the deposited S. aureus ISP3) as a
template. PCR is carried out under routine conditions, for
instance, in 25 .mu.l of reaction mixture with 0.5 .mu.g of the
above DNA template. A convenient reaction mixture is 1.5-5 mM
MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP, dCTP, dGTP,
dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five cycles of PCR (denaturation at 94.degree. C. for 1 min;
annealing at 55.degree. C. for 1 min; elongation at 72.degree. C.
for 1 min) are performed with a Perkin-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and the DNA band with expected molecular weight is
excised and purified. The PCR product is verified to be the
selected sequence by subcloning and sequencing the DNA product.
[0363] Finally, overlapping oligos of the DNA sequences of Table 1
can be synthesized and used to generate a nucleotide sequence of
desired length using PCR methods known in the art.
Example 2(a)
Expression and Purification staphylococcal polypeptides in E.
coli
[0364] The bacterial expression vector pQE60 is used for bacterial
expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic
resistance ("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin (QIAGEN, Inc., supra) and suitable single
restriction enzyme cleavage sites. These elements are arranged such
that an inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times.His tag")
covalently linked to the carboxyl terminus of that polypeptide.
[0365] The DNA sequence encoding the desired portion of a S. aureus
protein of the present invention is amplified from S. aureus
genomic DNA or from the deposited DNA clone using PCR
oligonucleotide primers which anneal to the 5' and 3' sequences
coding for the portion of the S. aureus polynucleotide. Additional
nucleotides containing restriction sites to facilitate cloning in
the pQE60 vector are added to the 5' and 3' sequences,
respectively.
[0366] For cloning the mature protein, the 5' primer has a sequence
containing an appropriate restriction site followed by nucleotides
of the amino terminal coding sequence of the desired S. aureus
polynucleotide sequence in Table 1. One of ordinary skill in the
art would appreciate that the point in the protein coding sequence
where the 5' and 3' primers begin may be varied to amplify a DNA
segment encoding any desired portion of the complete protein
shorter or longer than the mature form. The 3' primer has a
sequence containing an appropriate restriction site followed by
nucleotides complementary to the 3' end of the desired coding
sequence of Table 1, excluding a stop codon, with the coding
sequence aligned with the restriction site so as to maintain its
reading frame with that of the six His codons in the pQE60
vector.
[0367] The amplified S. aureus DNA fragment and the vector pQE60
are digested with restriction enzymes which recognize the sites in
the primers and the digested DNAs are then ligated together. The S.
aureus DNA is inserted into the restricted pQE60 vector in a manner
which places the S. aureus protein coding region downstream from
the IPTG-inducible promoter and in-frame with an initiating AUG and
the six histidine codons.
[0368] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al., supra. E. coli strain M15/rep4, containing multiple copies
of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which is only
one of many that are suitable for expressing a S. aureus
polypeptide, is available commercially (QIAGEN, Inc., supra).
Transformants are identified by their ability to grow on LB plates
in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0369] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") is then added to
a final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0370] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the S. aureus
polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (QIAGEN, Inc., supra). Proteins
with a 6.times.His tag bind to the Ni-NTA resin with high affinity
and can be purified in a simple one-step procedure (for details
see: The QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the
supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCl, pH
8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the S. aureus polypeptide is eluted with 6 M guanidine-HCl,
pH 5.
[0371] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. C.
[0372] Alternatively, the polypeptides of the present invention can
be produced by a non-denaturing method. In this method, after the
cells are harvested by centrifugation, the cell pellet from each
liter of culture is resuspended in 25 ml of Lysis Buffer A at
4.degree. C. (Lysis Buffer A=50 mM Na-phosphate, 300 mM NaCl, 10 mM
2-mercaptoethanol, 10% Glycerol, pH 7.5 with 1 tablet of Complete
EDTA-free protease inhibitor cocktail (Boehringer Mannheim
#1873580) per 50 ml of buffer). Absorbance at 550 nm is
approximately 10-20 O.D./ml. The suspension is then put through
three freeze/thaw cycles from -70.degree. C. (using a ethanol-dry
ice bath) up to room temperature. The cells are lysed via
sonication in short 10 sec bursts over 3 minutes at approximately
80W while kept on ice. The sonicated sample is then centrifuged at
15,000 RPM for 30 minutes at 4.degree. C. The supernatant is passed
through a column containing 1.0 ml of CL-4B resin to pre-clear the
sample of any proteins that may bind to agarose non-specifically,
and the flow-through fraction is collected.
[0373] The pre-cleared flow-through is applied to a
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(Quiagen, Inc., supra). Proteins with a 6.times.His tag bind to the
Ni-NTA resin with high affinity and can be purified in a simple
one-step procedure. Briefly, the supernatant is loaded onto the
column in Lysis Buffer A at 4.degree. C., the column is first
washed with 10 volumes of Lysis Buffer A until the A280 of the
eluate returns to the baseline. Then, the column is washed with 5
volumes of 40 mM Imidazole (92% Lysis Buffer A/8% Buffer B) (Buffer
B=50 mM Na-Phosphate, 300 mM NaCl, 10% Glycerol, 10 mM
2-mercaptoethanol, 500 mM Imidazole, pH of the final buffer should
be 7.5). The protein is eluted off of the column with a series of
increasing Imidazole solutions made by adjusting the ratios of
Lysis Buffer A to Buffer B. Three different concentrations are
used: 3 volumes of 75 mM Imidazole, 3 volumes of 150 mM Imidazole,
5 volumes of 500 mM Imidazole. The fractions containing the
purified protein are analyzed using 8%, 10% or 14% SDS-PAGE
depending on the protein size. The purified protein is then
dialyzed 2.times.against phosphate-buffered saline (PBS) in order
to place it into an easily workable buffer. The purified protein is
stored at 4.degree. C. or frozen at -800
[0374] The following is another alternative method may be used to
purify S. aureus expressed in E coli when it is present in the form
of inclusion bodies. Unless otherwise specified, all of the
following steps are conducted at 4-10C.
[0375] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0376] The cells are then lysed by passing the solution through a
microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0377] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 24 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the S. aureus polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0378] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0379] To clarify the refolded S. aureus polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0380] Fractions containing the S. aureus polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the S. aureus polypeptide (determined, for instance, by
16% SDS-PAGE) are then pooled.
[0381] The resultant S. aureus polypeptide exhibits greater than
95% purity after the above refolding and purification steps. No
major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
Example 2(b)
Expression and Purification staphylococcal polypeptides in E.
coli
[0382] Alternatively, the vector pQE10 can be used to clone and
express polypeptides of the present invention. The difference being
such that an inserted DNA fragment encoding a polypeptide expresses
that polypeptide with the six His residues (i.e., a "6.times.His
tag") covalently linked to the amino terminus of that polypeptide.
The bacterial expression vector pQE10 (QIAGEN, Inc., 9259 Eton
Avenue, Chatsworth, Calif., 91311) is used in this example. The
components of the pQE10 plasmid are arranged such that the inserted
DNA sequence encoding a polypeptide of the present invention
expresses the polypeptide with the six His residues (i.e., a
"6.times.His tag")) covalently linked to the amino terminus.
[0383] The DNA sequences encoding the desired portions of a
polypeptide of Table 1 are amplified using PCR oligonucleotide
primers from either genomic S. aureus DNA or DNA from the plasmid
clones listed in Table 1 clones of the present invention. The PCR
primers anneal to the nucleotide sequences encoding the desired
amino acid sequence of a polypeptide of the present invention.
Additional nucleotides containing restriction sites to facilitate
cloning in the pQE10 vector are added to the 5' and 3' primer
sequences, respectively.
[0384] For cloning a polypeptide of the present invention, the 5'
and 3' primers are selected to amplify their respective nucleotide
coding sequences. One of ordinary skill in the art would appreciate
that the point in the protein coding sequence where the 5' and 3'
primers begins may be varied to amplify a DNA segment encoding any
desired portion of a polypeptide of the present invention. The 5'
primer is designed so the coding sequence of the 6.times.His tag is
aligned with the restriction site so as to maintain its reading
frame with that of S. aureus polypeptide. The 3' is designed to
include an stop codon. The amplified DNA fragment is then cloned,
and the protein expressed, as described above for the pQE60
plasmid.
[0385] The DNA sequences encoding the amino acid sequences of Table
1 may also be cloned and expressed as fusion proteins by a protocol
similar to that described directly above, wherein the pET-32b(+)
vector (Novagen, 601 Science Drive, Madison, Wis. 53711) is
preferentially used in place of pQE10.
Example 2(c)
Expression and Purification of Staphylococcus polypeptides in E.
coli
[0386] The bacterial expression vector pQE60 is used for bacterial
expression in this example (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). However, in this example, the
polypeptide coding sequence is inserted such that translation of
the six His codons is prevented and, therefore, the polypeptide is
produced with no 6.times.His tag.
[0387] The DNA sequence encoding the desired portion of the S.
aureus amino acid sequence is amplified from a S. aureus genomic
DNA prep using PCR oligonucleotide primers which anneal to the 5'
and 3' nucleotide sequences corresponding to the desired portion of
the S. aureus polypeptides. Additional nucleotides containing
restriction sites to facilitate cloning in the pQE60 vector are
added to the 5' and 3' primer sequences.
[0388] For cloning a S. aureus polypeptides of the present
invention, 5' and 3' primers are selected to amplify their
respective nucleotide coding sequences. One of ordinary skill in
the art would appreciate that the point in the protein coding
sequence where the 5' and 3' primers begin may be varied to amplify
a DNA segment encoding any desired portion of a polypeptide of the
present invention. The 3' and 5' primers contain appropriate
restriction sites followed by nucleotides complementary to the 5'
and 3' ends of the coding sequence respectively. The 3' primer is
additionally designed to include an in-frame stop codon.
[0389] The amplified S. aureus DNA fragments and the vector pQE60
are digested with restriction enzymes recognizing the sites in the
primers and the digested DNAs are then ligated together. Insertion
of the S. aureus DNA into the restricted pQE60 vector places the S.
aureus protein coding region including its associated stop codon
downstream from the IPTG-inducible promoter and in-frame with an
initiating AUG. The associated stop codon prevents translation of
the six histidine codons downstream of the insertion point.
[0390] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al. E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which is only
one of many that are suitable for expressing S. aureus polypeptide,
is available commercially (QIAGEN, Inc., supra). Transformants are
identified by their ability to grow on LB plates in the presence of
ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
[0391] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0392] To purify the S. aureus polypeptide, the cells are then
stirred for 3-4 hours at 4.degree. C. in 6M guanidine-HCl, pH 8.
The cell debris is removed by centrifugation, and the supernatant
containing the S. aureus polypeptide is dialyzed against 50 mM
Na-acetate buffer pH 6, supplemented with 200 mM NaCl.
Alternatively, the protein can be successfully refolded by
dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH
7.4, containing protease inhibitors. After renaturation the protein
can be purified by ion exchange, hydrophobic interaction and size
exclusion chromatography. Alternatively, an affinity chromatography
step such as an antibody column can be used to obtain pure S.
aureus polypeptide. The purified protein is stored at 4.degree. C.
or frozen at -80.degree. C.
[0393] The following alternative method may be used to purify S.
aureus polypeptides expressed in E coli when it is present in the
form of inclusion bodies. Unless otherwise specified, all of the
following steps are conducted at 4-10.degree. C.
[0394] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0395] The cells ware then lysed by passing the solution through a
microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0396] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the S. aureus polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0397] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0398] To clarify the refolded S. aureus polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0399] Fractions containing the S. aureus polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the S. aureus polypeptide (determined, for instance, by
16% SDS-PAGE) are then pooled.
[0400] The resultant S. aureus polypeptide exhibits greater than
95% purity after the above refolding and purification steps. No
major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
Example 2(d)
Cloning and Expression of S. aureus in Other Bacteria
[0401] S. aureus polypeptides also can be produced in: S. aureus
using the methods of S. Skinner et al., (1988) Mol. Microbiol.
2:289-297 or J. I. Moreno (1996) Protein Expr. Purif. 8(3):332-340;
Lactobacillus using the methods of C. Rush et al., 1997 Appl.
Microbiol. Biotechnol. 47(5):537-542; or in Bacillus subtilis using
the methods Chang et al., U.S. Pat. No. 4,952,508.
Example 3
Cloning and Expression in COS Cells
[0402] A S. aureus expression plasmid is made by cloning a portion
of the DNA encoding a S. aureus polypeptide into the expression
vector pDNAI/Amp or pDNARIII (which can be obtained from
Invitrogen, Inc.). The expression vector pDNAI/amp contains: (1) an
E. coli origin of replication effective for propagation in E. coli
and other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a DNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al. 1984 Cell 37:767. The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pDNAIII contains, in addition, the selectable neomycin
marker.
[0403] A DNA fragment encoding a S. aureus polypeptide is cloned
into the polylinker region of the vector so that recombinant
protein expression is directed by the CMV promoter. The plasmid
construction strategy is as follows. The DNA from a S. aureus
genomic DNA prep is amplified using primers that contain convenient
restriction sites, much as described above for construction of
vectors for expression of S. aureus in E. coli. The 5' primer
contains a Kozak sequence, an AUG start codon, and nucleotides of
the 5' coding region of the S. aureus polypeptide. The 3' primer,
contains nucleotides complementary to the 3' coding sequence of the
S. aureus DNA, a stop codon, and a convenient restriction site.
[0404] The PCR amplified DNA fragment and the vector, pDNAI/Amp,
are digested with appropriate restriction enzymes and then ligated.
The ligation mixture is transformed into an appropriate E. coli
strain such as SURE.TM. (Stratagene Cloning Systems, La Jolla,
Calif. 92037), and the transformed culture is plated on ampicillin
media plates which then are incubated to allow growth of ampicillin
resistant colonies. Plasmid DNA is isolated from resistant colonies
and examined by restriction analysis or other means for the
presence of the fragment encoding the S. aureus polypeptide
[0405] For expression of a recombinant S. aureus polypeptide, COS
cells are transfected with an expression vector, as described
above, using DEAE-dextran, as described, for instance, by Sambrook
et al. (supra). Cells are incubated under conditions for expression
of S. aureus by the vector.
[0406] Expression of the S. aureus-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow et al., supra. To this end, two days after
transfection, the cells are labeled by incubation in media
containing .sup.35S-cysteine for 8 hours. The cells and the media
are collected, and the cells are washed and the lysed with
detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS,
1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et
al. (supra). Proteins are precipitated from the cell lysate and
from the culture media using an HA-specific monoclonal antibody.
The precipitated proteins then are analyzed by SDS-PAGE and
autoradiography. An expression product of the expected size is seen
in the cell lysate, which is not seen in negative controls.
Example 4
Cloning and Expression in CHO Cells
[0407] The vector pC4 is used for the expression of S. aureus
polypeptide in this example. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains
the mouse DHFR gene under control of the SV40 early promoter.
Chinese hamster ovary cells or other cells lacking dihydrofolate
activity that are transfected with these plasmids can be selected
by growing the cells in a selective medium (alpha minus MEM, Life
Technologies) supplemented with the chemotherapeutic agent
methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented. See,
e.g., Alt et al., 1978, J. Biol. Chem. 253:1357-1370; Hamlin et
al., 1990, Biochem. et Biophys. Acta, 1097:107-143; Page et al.,
1991, Biotechnology 9:64-68. Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[0408] Plasmid pC4 contains the strong promoter of the long
terminal repeat (LTR) of the Rouse Sarcoma Virus, for expressing a
polypeptide of interest, Cullen, et al. (1985) Mol. Cell. Biol.
5:438-447; plus a fragment isolated from the enhancer of the
immediate early gene of human cytomegalovirus (CMV), Boshart, et
al., 1985, Cell 41:521-530. Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: Bam HI, Xba I, and Asp 718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human B-actin promoter, the SV40 early or late promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
Clontech's Tet-Off and Tet-On gene expression systems and similar
systems can be used to express the S. aureus polypeptide in a
regulated way in mammalian cells (Gossen et al., 1992, Proc. Natl.
Acad. Sci. USA 89:5547-5551. For the polyadenylation of the mRNA
other signals, e.g., from the human growth hormone or globin genes
can be used as well. Stable cell lines carrying a gene of interest
integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418 or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[0409] The plasmid pC4 is digested with the restriction enzymes and
then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel. The DNA sequence encoding the S. aureus polypeptide is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the desired portion of the gene. A 5' primer
containing a restriction site, a Kozak sequence, an AUG start
codon, and nucleotides of the 5' coding region of the S. aureus
polypeptide is synthesized and used. A 3' primer, containing a
restriction site, stop codon, and nucleotides complementary to the
3' coding sequence of the S. aureus polypeptides is synthesized and
used. The amplified fragment is digested with the restriction
endonucleases and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0410] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using a
lipid-mediated transfection agent such as Lipofectin.TM. or
LipofectAMINE..TM. (LifeTechnologies Gaithersburg, Md.). The
plasmid pSV2-neo contains a dominant selectable marker, the neo
gene from Tn5 encoding an enzyme that confers resistance to a group
of antibiotics including G418. The cells are seeded in alpha minus
MEM supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of methotrexate plus 1 mg/ml G418. After about 10.sup.-14 days
single clones are trypsinized and then seeded in 6-well petri
dishes or 10 ml flasks using different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 5
Quantitative Murine Soft Tissue Infection Model for S. aureus
[0411] Compositions of the present invention, including
polypeptides and peptides, are assayed for their ability to
function as vaccines or to enhance/stimulate an immune response to
a bacterial species (e.g., S. aureus) using the following
quantitative murine soft tissue infection model. Mice (e.g., NIH
Swiss female mice, approximately 7 weeks old) are first treated
with a biologically protective effective amount, or immune
enhancing/stimulating effective amount of a composition of the
present invention using methods known in the art, such as those
discussed above. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). An
example of an appropriate starting dose is 20 .mu.g per animal.
[0412] The desired bacterial species used to challenge the mice,
such as S. aureus, is grown as an overnight culture. The culture is
diluted to a concentration of 5.times.10.sup.8 cfu/ml, in an
appropriate media, mixed well, serially diluted, and titered. The
desired doses are further diluted 1:2 with sterilized Cytodex 3
microcarrier beads preswollen in sterile PBS (3 g/100 ml). Mice are
anesthetize briefly until docile, but still mobile and injected
with 0.2 ml of the Cytodex 3 bead/bacterial mixture into each
animal subcutaneously in the inguinal region. After four days,
counting the day of injection as day one, mice are sacrificed and
the contents of the abscess is excised and placed in a 15 ml
conical tube containing 1.0 ml of sterile PBS. The contents of the
abscess is then enzymatically treated and plated as follows.
[0413] The abscess is first disrupted by vortexing with sterilized
glass beads placed in the tubes. 3.0 mls of prepared enzyme mixture
(1.0 mi Collagenase D (4.0 mg/ml), 1.0 ml Trypsin (6.0 mg/ml) and
8.0 ml PBS) is then added to each tube followed by a 20 min.
incubation at 37 C. The solution is then centrifuged and the
supernatant drawn off. 0.5 ml dH2O is then added and the tubes are
vortexed and then incubated for 10 min. at room temperature. 0.5 ml
media is then added and samples are serially diluted and plated
onto agar plates, and grown overnight at 37 C. Plates with distinct
and separate colonies are then counted, compared to positive and
negative control samples, and quantified. The method can be used to
identify composition and determine appropriate and effective doses
for humans and other animals by comparing the effective doses of
compositions of the present invention with compositions known in
the art to be effective in both mice and humans. Doses for the
effective treatment of humans and other animals, using compositions
of the present invention, are extrapolated using the data from the
above experiments of mice. It is appreciated that further studies
in humans and other animals may be needed to determine the most
effective doses using methods of clinical practice known in the
art.
Example 6
Murine Systemic Neutropenic Model for S. aureus Infection
[0414] Compositions of the present invention, including
polypeptides and peptides, are assayed for their ability to
function as vaccines or to enhance/stimulate an immune response to
a bacterial species (e.g., S. aureus) using the following
qualitative murine systemic neutropenic model. In addition,
antibodies of the present invention are employed to provide passive
immune or immunophylatic therapy prior to or post S aureus
infection. Mice (e.g., NIH Swiss female mice, approximately 7 weeks
old) are first treated with a biologically protective effective
amount, or immune enhancing/stimulating effective amount of a
composition of the present invention using methods known in the
art, such as those discussed above. See, e.g., Harlow et al.,
ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988). An example of an appropriate starting dose is
20 .mu.g of protein per animal. Mice are then injected with 250-300
mg/kg cyclophosphamide intraperitonially. Counting the day of C.P.
injection as day one, the mice are left untreated for 5 days to
begin recovery of PMNL'S.
[0415] The desired bacterial species used to challenge the mice,
such as S. aureus, is grown as an overnight culture. The culture is
diluted to a concentration of 5.times.10.sup.8 cfu/ml, in an
appropriate media, mixed well, serially diluted, and titered. The
desired doses are further diluted 1:2 in 4% Brewer's yeast in
media.
[0416] Mice are injected with the bacteria/brewer's yeast challenge
intraperitonially. The Brewer's yeast solution alone is used as a
control. The mice are then monitored twice daily for the first week
following challenge, and once a day for the next week to ascertain
morbidity and mortality. Mice remaining at the end of the
experiment are sacrificed. The method can be used to identify
compositions and determine appropriate and effective doses for
humans and other animals by comparing the effective doses of
compositions of the present invention with compositions known in
the art to be effective in both mice and humans. Doses for the
effective treatment of humans and other animals, using compositions
of the present invention, are extrapolated using the data from the
above experiments of mice. It is appreciated that further studies
in humans and other animals may be needed to determine the most
effective doses using methods of clinical practice known in the
art.
Example 7
Murine Lethal Sepsis Model
[0417] S. aureus polypeptides of the present invention can be
evaluated for potential vaccine efficacy using the murine lethal
sepsis model. In this model, mice are challenged with low lethal
doses (for example, between 10.sup.6 and 10.sup.7 colony forming
units [cfu]) of virulent strains of S. aureus. Initial studies are
conducted to determine a less virulent yet lethal strain of S.
aureus to determine its LD.sub.50. Polypeptides of the present
invention (e.g., the polypeptides described in Table 1, fragments
thereof and fragments that comprise the epitopes shown in Table 4),
produced as Examples 2 a-d, 3 and 4, and optionally conjugated with
another immunogen, are tested as vaccine candidates. Vaccine
candidates are selected as protective antigens if they can protect
against death when approximately 100 times the LD.sub.50 of the
strain is employed. Immunized mice are then challenged with a
lethal dose of S. aureus.
[0418] More specifically, female C2H/HeJ mices are immunized
subcutaneously in groups of 10 with 15 .mu.g of the protein of the
present invention formulated in complete Freund's adjuvant (CFA).
Twenty one days later, mice are boosted in the same way with
protein formulated in incomplete Freund's adjuvant. Twenty-eight
days following the boost, animals are bled and immune titers
against S. aureus proteins are determined by ELISA. 35 days
following the boost, a freshly prepared culture of S. aureus in BHI
(Brain Heart Infusion) both is diluted to approximately 35 to
100.times.LD.sub.50 in sterile PBS. A lethal dose is then injected
intraperitoneally into mice in a volume of 100 .mu.l. Mice are
monitored for 14 days for mortality. Survival rate is compared with
a sham group immunized with PBS and adjuvant alone.
Example 8
Identifying Vaccine Antigens Prevalent in S. Aureus Strains
[0419] It is further determined whether the majority of the most
prevalent S. aureus strains express the vaccine antigen(s) and
polypeptide(s) identified by the lethal model of Example 7 or the
models of Examples 5 or 6. Immunoblot analysis is performed with
cell lysates prepared from Staphylococcus strains representative of
the major capsular serotypes and probed with polyclonal antisera
specific for the protective antigens. A preferred vaccine is
comprised of a serological epitope of the polypeptide of the
present invention that is well conserved among the majority of
infective Staphyloccus serotypes.
Example 9
Production of an Antibody
[0420] a) Hybridoma Technology
[0421] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing polypeptide(s) of the
invention are administered to an animal to induce the production of
sera containing polyclonal antibodies. In a preferred method, a
preparation of polypeptide(s) of the invention is prepared and
purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to
produce polyclonal antisera of greater specific activity.
[0422] Monoclonal antibodies specific for polypeptide(s) of the
invention are prepared using hybridoma technology. (Kohler et al.,
Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511
(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a
mouse) is immunized with polypeptide(s) of the invention or, more
preferably, with a secreted polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably in Eagle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 g/l of nonessential
amino acids, about 1,000 U/ml of penicillin, and about 100 .mu.g/ml
of streptomycin.
[0423] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP20), available
from the ATCC. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981)). The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the polypeptide(s) of the invention.
[0424] Alternatively, additional antibodies capable of binding to
polypeptide(s) of the invention can be produced in a two-step
procedure using anti-idiotypic antibodies. Such a method makes use
of the fact that antibodies are themselves antigens, and therefore,
it is possible to obtain an antibody which binds to a second
antibody. In accordance with this method, protein specific
antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma
cells, and the hybridoma cells are screened to identify clones
which produce an antibody whose ability to bind to the
protein-specific antibody can be blocked by polypeptide(s) of the
invention. Such antibodies comprise anti-idiotypic antibodies to
the protein-specific antibody and are used to immunize an animal to
induce formation of further protein-specific antibodies. For in
vivo use of antibodies in humans, an antibody is "humanized". Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric and humanized antibodies are
known in the art and are discussed herein. (See, for review,
Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214
(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al.,
EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature
312:643 (1984); Neuberger et al., Nature 314:268 (1985).)
[0425] b) Isolation Of Antibody Fragments Directed Against
Polypeptide(s) From A Library Of scFvs
[0426] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against polypeptide(s) of the invention to which the
donor may or may not have been exposed (see e.g., U.S. Pat. No.
5,885,793 incorporated herein by reference in its entirety).
[0427] Rescue of the Library
[0428] A library of scFvs is constructed from the RNA of human PBLs
as described in PCT publication WO 92/01047. To rescue phage
displaying antibody fragments, approximately 109 E. coli harboring
the phagemid are used to inoculate 50 ml of 2.times.TY containing
1% glucose and 100 .mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and
grown to an O.D. of 0.8 with shaking. Five ml of this culture is
used to innoculate 50 ml of 2.times.TY-AMP-GLU, 2.times.10.sup.8 TU
of delta gene 3 helper (M13 delta gene III, see PCT publication WO
92/01047) are added and the culture incubated at 37.degree. C. for
45 minutes without shaking and then at 37.degree. C. for 45 minutes
with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min.
and the pellet resuspended in 2 liters of 2.times.TY containing 100
.mu.g/ml ampicillin and 50 .mu.g/ml kanamycin and grown overnight.
Phage are prepared as described in PCT publication WO 92/01047.
[0429] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[0430] Panning of the Library
[0431] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 .mu.g/ml or 10 .mu.g/ml of a polypeptide of the present
invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at
37.degree. C. and then washed 3 times in PBS. Approximately 1013 TU
of phage is applied to the tube and incubated for 30 minutes at
room temperature tumbling on an over and under turntable and then
left to stand for another 1.5 hours. Tubes are washed 10 times with
PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding
1 ml of 100 mM triethylamine and rotating 15 minutes on an under
and over turntable after which the solution is immediately
neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then
used to infect 10 ml of mid-log E. coli TG1 by incubating eluted
phage with bacteria for 30 minutes at 37.degree. C. The E. coli are
then plated on TYE plates containing 1% glucose and 100 .mu.g/ml
ampicillin. The resulting bacterial library is then rescued with
delta gene 3 helper phage as described above to prepare phage for a
subsequent round of selection. This process is then repeated for a
total of 4 rounds of affinity purification with tube-washing
increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS
for rounds 3 and 4.
[0432] Characterization of Binders
[0433] Eluted phage from the 3rd and 4th rounds of selection are
used to infect E. coli HB 2151 and soluble scFv is produced (Marks,
et al., 1991) from single colonies for assay. ELISAs are performed
with microtitre plates coated with either 10 .mu.g/ml of the
polypeptide of the present invention in 50 mM bicarbonate pH 9.6.
Clones positive in ELISA are further characterized by PCR
fingerprinting (see, e.g., PCT publication WO 92/01047) and then by
sequencing. These ELISA positive clones may also be further
characterized by techniques known in the art, such as, for example,
epitope mapping, binding affinity, receptor signal transduction,
ability to block or competitively inhibit antibody/antigen binding,
and competitive agonistic or antagonistic activity.
[0434] The disclosure of all publications (including patents,
patent applications, journal articles, laboratory manuals, books,
or other documents) cited herein and the sequence listings are
hereby incorporated by reference in their entireties.
[0435] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention. Functionally
equivalent methods and components are within the scope of the
invention, in addition to those shown and described herein and will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 1
1
61 1 1092 DNA Staphylococcus aureus 1 attaactagt caatattcct
acctctgact tgagtttaaa aagtaatcta tgttaaatta 60 atacctggta
ttaaaaattt tattaagaag gtgttcaact atgaacgtgg gtattaaagg 120
ttttggtgca tatgcgccag aaaagattat tgacaatgcc tattttgagc aatttttaga
180 tacatctgat gaatggattt ctaagatgac tggaattaaa gaaagacatt
gggcagatga 240 tgatcaagat acttcagatt tagcatatga agcaagttta
aaagcaatcg ctgacgctgg 300 tattcagccc gaagatatag atatgataat
tgttgccaca gcaactggag atatgccatt 360 tccaactgtc gcaaatatgt
tgcaagaacg tttagggacg ggcaaagttg cctctatgga 420 tcaacttgca
gcatgttctg gatttatgta ttcaatgatt acagctaaac aatatgttca 480
atctggagat tatcataaca ttttagttgt cggtgcagat aaattatcta aaataacaga
540 tttaactgac cgttctactg cagttctatt tggagatggt gcaggtgcgg
ttatcatcgg 600 tgaagtttca gatggcagag gtattataag ttatgaaatg
ggttctgatg gcacaggtgg 660 taaacattta tatttagata aagatactgg
taaactgaaa atgaatggtc gagaagtatt 720 taaatttgct gttagaatta
tgggtgatgc atcaacacgt gtagttgaaa aagcgaattt 780 aacatcagat
gatatagatt tatttattcc tcatcaagct aatattagaa ttatggaatc 840
agctagagaa cgcttaggta tttcaaaaga caaaatgagt gtttctgtaa ataaatatgg
900 aaatacttca gctgcgtcaa tacctttaag tatcgatcaa gaattaaaaa
atggtaaaat 960 caaagatgat gatacaattg ttcttgtcgg attcggtggc
ggcctaactt ggggcgcaat 1020 gacaataaaa tggggaaaat aggaggataa
cgaatgagtc aaaataaaag agtagttatt 1080 acaggtatgg ga 1092 2 313 PRT
Staphylococcus aureus 2 Met Asn Val Gly Ile Lys Gly Phe Gly Ala Tyr
Ala Pro Glu Lys Ile 1 5 10 15 Ile Asp Asn Ala Tyr Phe Glu Gln Phe
Leu Asp Thr Ser Asp Glu Trp 20 25 30 Ile Ser Lys Met Thr Gly Ile
Lys Glu Arg His Trp Ala Asp Asp Asp 35 40 45 Gln Asp Thr Ser Asp
Leu Ala Tyr Glu Ala Ser Leu Lys Ala Ile Ala 50 55 60 Asp Ala Gly
Ile Gln Pro Glu Asp Ile Asp Met Ile Ile Val Ala Thr 65 70 75 80 Ala
Thr Gly Asp Met Pro Phe Pro Thr Val Ala Asn Met Leu Gln Glu 85 90
95 Arg Leu Gly Thr Gly Lys Val Ala Ser Met Asp Gln Leu Ala Ala Cys
100 105 110 Ser Gly Phe Met Tyr Ser Met Ile Thr Ala Lys Gln Tyr Val
Gln Ser 115 120 125 Gly Asp Tyr His Asn Ile Leu Val Val Gly Ala Asp
Lys Leu Ser Lys 130 135 140 Ile Thr Asp Leu Thr Asp Arg Ser Thr Ala
Val Leu Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Val Ile Ile Gly
Glu Val Ser Asp Gly Arg Gly Ile Ile 165 170 175 Ser Tyr Glu Met Gly
Ser Asp Gly Thr Gly Gly Lys His Leu Tyr Leu 180 185 190 Asp Lys Asp
Thr Gly Lys Leu Lys Met Asn Gly Arg Glu Val Phe Lys 195 200 205 Phe
Ala Val Arg Ile Met Gly Asp Ala Ser Thr Arg Val Val Glu Lys 210 215
220 Ala Asn Leu Thr Ser Asp Asp Ile Asp Leu Phe Ile Pro His Gln Ala
225 230 235 240 Asn Ile Arg Ile Met Glu Ser Ala Arg Glu Arg Leu Gly
Ile Ser Lys 245 250 255 Asp Lys Met Ser Val Ser Val Asn Lys Tyr Gly
Asn Thr Ser Ala Ala 260 265 270 Ser Ile Pro Leu Ser Ile Asp Gln Glu
Leu Lys Asn Gly Lys Ile Lys 275 280 285 Asp Asp Asp Thr Ile Val Leu
Val Gly Phe Gly Gly Gly Leu Thr Trp 290 295 300 Gly Ala Met Thr Ile
Lys Trp Gly Lys 305 310 3 1074 DNA Staphylococcus aureus 3
atactaattc taatactttc ttttcaattt tcgcaaatga attttaaaat tggtataata
60 ctatatgata ttaaagacat gagaaaggat gtactgagaa gtgataaata
aagacatcta 120 tcaagcttta caacaactta tcccaaatga aaaaattaaa
gttgatgaac ctttaaaacg 180 atacacttat actaaaacag gtggtaatgc
cgacttttac attaccccta ctaaaaatga 240 agaagtacaa gcagttgtta
aatatgccta tcaaaatgag attcctgtta catatttagg 300 aaatggctca
aatattatta tccgtgaagg tggtattcgc ggtattgtaa ttagtttatt 360
atcactagat catatcgaag tatctgatga tgcgataata gccggtagcg gcgctgcaat
420 tattgatgtc tcacgtgttg ctcgtgatta cgcacttact ggccttgaat
ttgcatgtgg 480 tattccaggt tcaattggtg gtgcagtgta tatgaatgct
ggcgcttatg gtggcgaagt 540 taaagattgt atagactatg cgctttgcgt
aaacgaacaa ggctcgttaa ttaaacttac 600 aacaaaagaa ttagagttag
attatcgtaa tagcattatt caaaaagaac acttagttgt 660 attagaagct
gcatttactt tagctcctgg taaaatgact gaaatacaag ctaaaatgga 720
tgatttaaca gaacgtagag aatctaaaca acctttagag tatccttcat gtggtagtgt
780 attccaaaga ccgcctggtc attttgcagg taaattgata caagattcta
atttgcaagg 840 tcaccgtatt ggcggcgttg aagtttcaac caaacacgct
ggttttatgg taaatgtaga 900 caatggaact gctacagatt atgaaaacct
tattcattat gtacaaaaga ccgtcaaaga 960 aaaatttggc attgaattaa
atcgtgaagt tcgcattatt ggtgaacatc caaaggaatc 1020 gtaagttaag
gagctttgtc tatgcctaaa gtttatggtt cattaatcga tact 1074 4 307 PRT
Staphylococcus aureus 4 Val Ile Asn Lys Asp Ile Tyr Gln Ala Leu Gln
Gln Leu Ile Pro Asn 1 5 10 15 Glu Lys Ile Lys Val Asp Glu Pro Leu
Lys Arg Tyr Thr Tyr Thr Lys 20 25 30 Thr Gly Gly Asn Ala Asp Phe
Tyr Ile Thr Pro Thr Lys Asn Glu Glu 35 40 45 Val Gln Ala Val Val
Lys Tyr Ala Tyr Gln Asn Glu Ile Pro Val Thr 50 55 60 Tyr Leu Gly
Asn Gly Ser Asn Ile Ile Ile Arg Glu Gly Gly Ile Arg 65 70 75 80 Gly
Ile Val Ile Ser Leu Leu Ser Leu Asp His Ile Glu Val Ser Asp 85 90
95 Asp Ala Ile Ile Ala Gly Ser Gly Ala Ala Ile Ile Asp Val Ser Arg
100 105 110 Val Ala Arg Asp Tyr Ala Leu Thr Gly Leu Glu Phe Ala Cys
Gly Ile 115 120 125 Pro Gly Ser Ile Gly Gly Ala Val Tyr Met Asn Ala
Gly Ala Tyr Gly 130 135 140 Gly Glu Val Lys Asp Cys Ile Asp Tyr Ala
Leu Cys Val Asn Glu Gln 145 150 155 160 Gly Ser Leu Ile Lys Leu Thr
Thr Lys Glu Leu Glu Leu Asp Tyr Arg 165 170 175 Asn Ser Ile Ile Gln
Lys Glu His Leu Val Val Leu Glu Ala Ala Phe 180 185 190 Thr Leu Ala
Pro Gly Lys Met Thr Glu Ile Gln Ala Lys Met Asp Asp 195 200 205 Leu
Thr Glu Arg Arg Glu Ser Lys Gln Pro Leu Glu Tyr Pro Ser Cys 210 215
220 Gly Ser Val Phe Gln Arg Pro Pro Gly His Phe Ala Gly Lys Leu Ile
225 230 235 240 Gln Asp Ser Asn Leu Gln Gly His Arg Ile Gly Gly Val
Glu Val Ser 245 250 255 Thr Lys His Ala Gly Phe Met Val Asn Val Asp
Asn Gly Thr Ala Thr 260 265 270 Asp Tyr Glu Asn Leu Ile His Tyr Val
Gln Lys Thr Val Lys Glu Lys 275 280 285 Phe Gly Ile Glu Leu Asn Arg
Glu Val Arg Ile Ile Gly Glu His Pro 290 295 300 Lys Glu Ser 305 5
916 DNA Staphylococcus aureus 5 aatagtgtta aaatgtattg acgaataaaa
agttagttaa aactgggatt agatattcta 60 tccgttaaat taattattat
aaggagttat cttacatgtt aaatcttgaa aacaaaacat 120 atgtcatcat
gggaatcgct aataagcgta gtattgcttt tggtgtcgct aaagttttag 180
atcaattagg tgctaaatta gtatttactt accgtaaaga acgtagccgt aaagagcttg
240 aaaaattatt agaacaatta aatcaaccag aagcgcactt atatcaaatt
gatgttcaaa 300 gcgatgaaga ggttattaat ggttttgagc aaattggtaa
agatgttggc aatattgatg 360 gtgtatatca ttcaatcgca tttgctaata
tggaagactt acgcggacgc ttttctgaaa 420 cttcacgtga aggcttcttg
ttagctcaag acattagttc ttactcatta acaattgtgg 480 ctcatgaagc
taaaaaatta atgccagaag gtggtagcat tgttgcaaca acatatttag 540
gtggcgaatt cgcagttcaa aactataatg tgatgggtgt tgctaaagcg agcttagaag
600 caaatgttaa atatttagca ttagacttag gtccagataa tattcgcgtt
aatgcaattt 660 cagctagtcc aatccgtaca ttaagtgcaa aaggtgtggg
tggtttcaat acaattctta 720 aagaaatcga agagcgtgca cctttaaaac
gtaatgttga tcaagtagaa gtaggtaaaa 780 ctgcggctta cttattaagt
gatttatcaa gtggcgttac aggtgaaaat attcatgtag 840 atagcggatt
ccacgcaatt aaataatatc attcaacagc tttgttcacg ttattatata 900
tgtgagcaaa gctttt 916 6 256 PRT Staphylococcus aureus 6 Met Leu Asn
Leu Glu Asn Lys Thr Tyr Val Ile Met Gly Ile Ala Asn 1 5 10 15 Lys
Arg Ser Ile Ala Phe Gly Val Ala Lys Val Leu Asp Gln Leu Gly 20 25
30 Ala Lys Leu Val Phe Thr Tyr Arg Lys Glu Arg Ser Arg Lys Glu Leu
35 40 45 Glu Lys Leu Leu Glu Gln Leu Asn Gln Pro Glu Ala His Leu
Tyr Gln 50 55 60 Gly Lys Asp Val Gly Asn Ile Asp Gly Val Tyr His
Ser Ile Ala Phe 65 70 75 80 Ile Asp Val Gln Ser Asp Glu Glu Val Ile
Asn Gly Phe Glu Gln Ile 85 90 95 Ala Asn Met Glu Asp Leu Arg Gly
Arg Phe Ser Glu Thr Ser Arg Glu 100 105 110 Gly Phe Leu Leu Ala Gln
Asp Ile Ser Ser Tyr Ser Leu Thr Ile Val 115 120 125 Ala His Glu Ala
Lys Lys Leu Met Pro Glu Gly Gly Ser Ile Val Ala 130 135 140 Thr Thr
Tyr Leu Gly Gly Glu Phe Ala Val Gln Asn Tyr Asn Val Met 145 150 155
160 Gly Val Ala Lys Ala Ser Leu Glu Ala Asn Val Lys Tyr Leu Ala Leu
165 170 175 Asp Leu Gly Pro Asp Asn Ile Arg Val Asn Ala Ile Ser Ala
Ser Pro 180 185 190 Ile Arg Thr Leu Ser Ala Lys Gly Val Gly Gly Phe
Asn Thr Ile Leu 195 200 205 Lys Glu Ile Glu Glu Arg Ala Pro Leu Lys
Arg Asn Val Asp Gln Val 210 215 220 Glu Val Gly Lys Thr Ala Ala Tyr
Leu Leu Ser Asp Leu Ser Ser Gly 225 230 235 240 Val Thr Gly Glu Asn
Ile His Val Asp Ser Gly Phe His Ala Ile Lys 245 250 255 7 1376 DNA
Staphylococcus aureus 7 taaaataatt ttaaaatagg gaaatgtaaa gtaataggag
ttctaagtgg aggatttacg 60 atggataaaa tagtaatcaa aggtggaaat
aaattaacgg gtgaagttaa agtagaaggt 120 gctaaaaatg cagtattacc
aatattgaca gcatctttat tagcttctga taaaccgagc 180 aaattagtta
atgttccagc tttaagtgat gtagaaacaa taaataatgt attaacaact 240
ttaaatgctg acgttacata caaaaaggac gaaaatgctg ttgtcgttga tgcaacaaag
300 actctaaatg aagaggcacc atatgaatat gttagtaaaa tgcgtgcaag
tattttagtt 360 atgggacctc ttttagcaag actaggacat gctattgttg
cattgcctgg tggttgtgca 420 attggaagta gaccgattga gcaacacatt
aaaggttttg aagctttagg cgcagaaatt 480 catcttgaaa atggtaatat
ttatgctaat gctaaagatg gattaaaagg tacatcaatt 540 catttagatt
ttccaagtgt aggagcaaca caaaatatta ttatggcagc atcattagct 600
aagggtaaga ctttaattga aaatgcagct aaagaacctg aaattgtcga tttagcaaac
660 tacattaatg aaatgggtgg tagaattact ggtgctggta cagacacaat
tacaatcaat 720 ggtgtagaat cattacatgg tgtagaacat gctatcattc
cagatagaat tgaagcaggc 780 acattactaa tcgctggtgc tataacgcgt
ggtgatattt ttgtacgtgg tgcaatcaaa 840 gaacatatgg cgagtttagt
ctataaacta gaagaaatgg gcgttgaatt ggactatcaa 900 gaagatggta
ttcgtgtacg tgctgaaggg gaattacaac ctgtagacat caaaactcta 960
ccacatcctg gattcccgac tgatatgcaa tcacaaatga tggcattgtt attaacggca
1020 aatggtcata aagtcgtaac cgaaactgtt tttgaaaacc gttttatgca
tgttgcagag 1080 ttcaaacgta tgaatgctaa tatcaatgta gaaggtcgta
gtgctaaact tgaaggtaaa 1140 agtcaattgc aaggtgcaca agttaaagcg
actgatttaa gagcagcagc cgccttaatt 1200 ttagctggat tagttgctga
tggtaaaaca agcgttactg aattaacgca cctagataga 1260 ggctatgttg
acttacacgg taaattgaag caattaggtg cagacattga acgtattaac 1320
gattaattca gtaaattaat ataatggagg atttcaacca tggaaacaat ttttga 1376
8 421 PRT Staphylococcus aureus 8 Met Asp Lys Ile Val Ile Lys Gly
Gly Asn Lys Leu Thr Gly Glu Val 1 5 10 15 Lys Val Glu Gly Ala Lys
Asn Ala Val Leu Pro Ile Leu Thr Ala Ser 20 25 30 Leu Leu Ala Ser
Asp Lys Pro Ser Lys Leu Val Asn Val Pro Ala Leu 35 40 45 Ser Asp
Val Glu Thr Ile Asn Asn Val Leu Thr Thr Leu Asn Ala Asp 50 55 60
Val Thr Tyr Lys Lys Asp Glu Asn Ala Val Val Val Asp Ala Thr Lys 65
70 75 80 Thr Leu Asn Glu Glu Ala Pro Tyr Glu Tyr Val Ser Lys Met
Arg Ala 85 90 95 Ser Ile Leu Val Met Gly Pro Leu Leu Ala Arg Leu
Gly His Ala Ile 100 105 110 Val Ala Leu Pro Gly Gly Cys Ala Ile Gly
Ser Arg Pro Ile Glu Gln 115 120 125 His Ile Lys Gly Phe Glu Ala Leu
Gly Ala Glu Ile His Leu Glu Asn 130 135 140 Gly Asn Ile Tyr Ala Asn
Ala Lys Asp Gly Leu Lys Gly Thr Ser Ile 145 150 155 160 His Leu Asp
Phe Pro Ser Val Gly Ala Thr Gln Asn Ile Ile Met Ala 165 170 175 Ala
Ser Leu Ala Lys Gly Lys Thr Leu Ile Glu Asn Ala Ala Lys Glu 180 185
190 Pro Glu Ile Val Asp Leu Ala Asn Tyr Ile Asn Glu Met Gly Gly Arg
195 200 205 Ile Thr Gly Ala Gly Thr Asp Thr Ile Thr Ile Asn Gly Val
Glu Ser 210 215 220 Leu His Gly Val Glu His Ala Ile Ile Pro Asp Arg
Ile Glu Ala Gly 225 230 235 240 Thr Leu Leu Ile Ala Gly Ala Ile Thr
Arg Gly Asp Ile Phe Val Arg 245 250 255 Gly Ala Ile Lys Glu His Met
Ala Ser Leu Val Tyr Lys Leu Glu Glu 260 265 270 Met Gly Val Glu Leu
Asp Tyr Gln Glu Asp Gly Ile Arg Val Arg Ala 275 280 285 Glu Gly Glu
Leu Gln Pro Val Asp Ile Lys Thr Leu Pro His Pro Gly 290 295 300 Phe
Pro Thr Asp Met Gln Ser Gln Met Met Ala Leu Leu Leu Thr Ala 305 310
315 320 Asn Gly His Lys Val Val Thr Glu Thr Val Phe Glu Asn Arg Phe
Met 325 330 335 His Val Ala Glu Phe Lys Arg Met Asn Ala Asn Ile Asn
Val Glu Gly 340 345 350 Arg Ser Ala Lys Leu Glu Gly Lys Ser Gln Leu
Gln Gly Ala Gln Val 355 360 365 Lys Ala Thr Asp Leu Arg Ala Ala Ala
Ala Leu Ile Leu Ala Gly Leu 370 375 380 Val Ala Asp Gly Lys Thr Ser
Val Thr Glu Leu Thr His Leu Asp Arg 385 390 395 400 Gly Tyr Val Asp
Leu His Gly Lys Leu Lys Gln Leu Gly Ala Asp Ile 405 410 415 Glu Arg
Ile Asn Asp 420 9 1537 DNA Staphylococcus aureus 9 ttcatgtatt
taaaaggttg gggattagca taatgggatt gtgctagcac agttatttat 60
gcattgtcat gcctatctat tacttactaa ctaaaaaata atgaaatggg tgtaaactat
120 atgcctgaaa gagaacgtac atctcctcag tatgaatcat tccacgaatt
gtacaagaac 180 tatactacca aggaactcac tcaaaaagct aaaactctta
agttgacgaa ccatagtaaa 240 ttaaataaaa aagaacttgt tctagctatt
atggaagcac aaatggaaaa agatggtaac 300 tattatatgg aaggtatctt
agatgatata caaccaggtg gttatggttt tttaagaaca 360 gtgaactatt
ctaaagggga aaaagatatt tatatatctg ctagccaaat tcgtcgtttt 420
gaaattaaac gtggggataa agtaactggg aaagttagaa aacctaaaga taacgaaaaa
480 tattatggct tattacaagt tgactttgtc aatgaccata acgcagaaga
agtgaagaaa 540 cgtccgcatt tccaagcttt gacaccactt tatccagatg
agcgtattaa attagagaca 600 gaaatacaaa attattcaac gcgcatcatg
gatttagtaa caccgattgg tttaggtcaa 660 cgtggtttaa tagtggcgcc
acctaaagca ggtaaaacat cgttattaaa agaaatagcg 720 aatgcaatca
gtacgaacaa accagatgca aagctattta ttttgttagt tggcgagcgt 780
cctgaagagg taacagattt agaacgctca gtagaagctg ctgaagtcgt tcattcaacg
840 tttgacgaac caccagaaca ccatgttaaa gtagctgaat tattacttga
acgtgcaaag 900 cgtttagtag aaattgggga agatgtcatt attttaatgg
attctataac gagattagca 960 cgcgcttata acttagttat tccaccaagt
ggtcgtacat tatcaggtgg tttagatcct 1020 gcatctttac acaaaccaaa
agcattcttc ggtgcagcga gaaatattga agcgggtgga 1080 agtttaacaa
tacttgcaac tgcattagtt gatacgggtt cacgtatgga cgatatgatt 1140
tacgaagaat ttaaaggaac aggtaacatg gagttacatt tagatcgtaa attgtctgaa
1200 cgtcgtatct tccctgcaat tgatattggc agaagttcaa cgcgtaaaga
agaattgttg 1260 ataagtaaat ctgaattaga cacattatgg caattaagaa
atctattcac tgactcaact 1320 gactttactg aaagatttat tcgcaaactt
aaaaggtcta agaataatga agatttcttc 1380 aagcagctac aaaagtctgc
agaagaaagt actaaaacgg gtcgacctat aatttaataa 1440 acattatata
ggggcttgcg ttttgaatta attaccttta taattacaca gtattgggta 1500
aaaactcaca aataactctg ttccagatgg ttcaggg 1537 10 438 PRT
Staphylococcus aureus 10 Met Pro Glu Arg Glu Arg Thr Ser Pro Gln
Tyr Glu Ser Phe His Glu 1 5 10 15 Leu Tyr Lys Asn Tyr Thr Thr Lys
Glu Leu Thr Gln Lys Ala Lys Thr 20 25 30 Leu Lys Leu Thr Asn His
Ser Lys Leu Asn Lys Lys Glu Leu Val Leu 35 40 45 Ala Ile Met Glu
Ala Gln Met Glu Lys Asp Gly Asn Tyr Tyr Met Glu 50 55 60 Gly Ile
Leu Asp Asp Ile Gln Pro Gly Gly Tyr Gly Phe Leu Arg Thr 65 70 75 80
Val Asn Tyr Ser Lys Gly Glu Lys Asp Ile Tyr Ile Ser Ala Ser Gln 85
90 95 Ile Arg Arg Phe Glu Ile Lys Arg Gly Asp Lys Val Thr Gly Lys
Val 100 105 110
Arg Lys Pro Lys Asp Asn Glu Lys Tyr Tyr Gly Leu Leu Gln Val Asp 115
120 125 Phe Val Asn Asp His Asn Ala Glu Glu Val Lys Lys Arg Pro His
Phe 130 135 140 Gln Ala Leu Thr Pro Leu Tyr Pro Asp Glu Arg Ile Lys
Leu Glu Thr 145 150 155 160 Glu Ile Gln Asn Tyr Ser Thr Arg Ile Met
Asp Leu Val Thr Pro Ile 165 170 175 Gly Leu Gly Gln Arg Gly Leu Ile
Val Ala Pro Pro Lys Ala Gly Lys 180 185 190 Thr Ser Leu Leu Lys Glu
Ile Ala Asn Ala Ile Ser Thr Asn Lys Pro 195 200 205 Asp Ala Lys Leu
Phe Ile Leu Leu Val Gly Glu Arg Pro Glu Glu Val 210 215 220 Thr Asp
Leu Glu Arg Ser Val Glu Ala Ala Glu Val Val His Ser Thr 225 230 235
240 Phe Asp Glu Pro Pro Glu His His Val Lys Val Ala Glu Leu Leu Leu
245 250 255 Glu Arg Ala Lys Arg Leu Val Glu Ile Gly Glu Asp Val Ile
Ile Leu 260 265 270 Met Asp Ser Ile Thr Arg Leu Ala Arg Ala Tyr Asn
Leu Val Ile Pro 275 280 285 Pro Ser Gly Arg Thr Leu Ser Gly Gly Leu
Asp Pro Ala Ser Leu His 290 295 300 Lys Pro Lys Ala Phe Phe Gly Ala
Ala Arg Asn Ile Glu Ala Gly Gly 305 310 315 320 Ser Leu Thr Ile Leu
Ala Thr Ala Leu Val Asp Thr Gly Ser Arg Met 325 330 335 Asp Asp Met
Ile Tyr Glu Glu Phe Lys Gly Thr Gly Asn Met Glu Leu 340 345 350 His
Leu Asp Arg Lys Leu Ser Glu Arg Arg Ile Phe Pro Ala Ile Asp 355 360
365 Ile Gly Arg Ser Ser Thr Arg Lys Glu Glu Leu Leu Ile Ser Lys Ser
370 375 380 Glu Leu Asp Thr Leu Trp Gln Leu Arg Asn Leu Phe Thr Asp
Ser Thr 385 390 395 400 Asp Phe Thr Glu Arg Phe Ile Arg Lys Leu Lys
Arg Ser Lys Asn Asn 405 410 415 Glu Asp Phe Phe Lys Gln Leu Gln Lys
Ser Ala Glu Glu Ser Thr Lys 420 425 430 Thr Gly Arg Pro Ile Ile 435
11 554 DNA Staphylococcus aureus 11 gatctttttt ttcgtttaaa
ttaagaataa atagaaattt atgttataag ctcaatagaa 60 gtttaaatat
agcttcaata aaaacgataa taagcgagtg atgttattgg aaaaagctta 120
ccgaattaaa aagaatgcag attttcagag aatatataaa aaaggtcatt ctgtagccaa
180 cagacaattt gttgtataca cttgtaataa taaagaaata gaccattttc
gcttaggtat 240 tagtgtttct aaaaaactag gtaatgcagt gttaagaaac
aagattaaaa gagcaatacg 300 tgaaaatttc aaagtacata agtcgcatat
attggccaaa gatattattg taatagcaag 360 acagccagct aaagatatga
cgactttaca aatacagaat agtcttgagc acgtacttaa 420 aattgccaaa
gtttttaata aaaagattaa gtaaggatag ggtaggggaa ggaaaacatt 480
aaccactcaa cacatcccga agtcttacct cagacaaacg taagactgac cttagggtta
540 taataactta cttt 554 12 117 PRT Staphylococcus aureus 12 Met Leu
Leu Glu Lys Ala Tyr Arg Ile Lys Lys Asn Ala Asp Phe Gln 1 5 10 15
Arg Ile Tyr Lys Lys Gly His Ser Val Ala Asn Arg Gln Phe Val Val 20
25 30 Tyr Thr Cys Asn Asn Lys Glu Ile Asp His Phe Arg Leu Gly Ile
Ser 35 40 45 Val Ser Lys Lys Leu Gly Asn Ala Val Leu Arg Asn Lys
Ile Lys Arg 50 55 60 Ala Ile Arg Glu Asn Phe Lys Val His Lys Ser
His Ile Leu Ala Lys 65 70 75 80 Asp Ile Ile Val Ile Ala Arg Gln Pro
Ala Lys Asp Met Thr Thr Leu 85 90 95 Gln Ile Gln Asn Ser Leu Glu
His Val Leu Lys Ile Ala Lys Val Phe 100 105 110 Asn Lys Lys Ile Lys
115 13 1712 DNA Staphylococcus aureus 13 cagcaaaaac tggtgaaggt
ggtaaattgt ttgggtcagt aagtacaaaa caaattgccg 60 aagcactaaa
agcacaacat gatattaaaa ttgataaacg taaaatggat ttaccaaatg 120
gaattcattc cctaggatat acgaatgtac ctgttaaatt agataaagaa gttgaaggta
180 caattcgcgt acacacagtt gaacaataaa gttggattga aataagaggt
gtaaccattc 240 atggatagaa tgtatgagca aaatcaaatg ccgcataaca
atgaagctga acagtctgtc 300 ttaggttcaa ttattataga tccagaattg
attaatacta ctcaggaagt tttgcttcct 360 gagtcgtttt ataggggtgc
ccatcaacat attttccgtg caatgatgca cttaaatgaa 420 gataataaag
aaattgatgt tgtaacattg atggatcaat tatcgacgga aggtacgttg 480
aatgaagcgg gtggcccgca atatcttgca gagttatcta caaatgtacc aacgacgcga
540 aatgttcagt attatactga tatcgtttct aagcatgcat taaaacgtag
attgattcaa 600 actgcagata gtattgccaa tgatggatat aatgatgaac
ttgaactaga tgcgatttta 660 agtgatgcag aacgtcgaat tttagagcta
tcatcttctc gtgaaagcga tggctttaaa 720 gacattcgag acgtcttagg
acaagtgtat gaaacagctg aagagcttga tcaaaatagt 780 ggtcaaacac
caggtatacc tacaggatat cgagatttag accaaatgac agcagggttc 840
aaccgaaatg atttaattat ccttgcagcg cgtccatctg taggtaagac tgcgttcgca
900 cttaatattg cacaaaaagt tgcaacgcat gaagatatgt atacagttgg
tattttctcg 960 ctagagatgg gtgctgatca gttagccaca cgtatgattt
gtagttctgg aaatgttgac 1020 tcaaaccgct taagaacggg tactatgact
gaggaagatt ggagtcgttt tactatagcg 1080 gtaggtaaat tatcacgtac
gaagattttt attgatgata caccgggtat tcgaattaat 1140 gatttacgtt
ctaaatgtcg tcgattaaag caagaacatg gcttagacat gattgtgatt 1200
gactacttac agttgattca aggtagtggt tcacgtgcgt ccgataacag acaacaggaa
1260 gtttctgaaa tctctcgtac attaaaagca ttagcccgtg aattaaaatg
tccagttatc 1320 gcattaagtc agttatctcg tggtgttgaa caacgacaag
ataaacgtcc aatgatgagt 1380 gatattcgtg aatctggttc gattgagcaa
gatgccgata tcgttgcatt cttataccgt 1440 gatgattact ataaccgtgg
cggcgatgaa gatgatgacg atgatggtgg tttcgagcca 1500 caaacgaatg
atgaaaacgg tgaaattgaa attatcattg ctaagcaacg taacggtcca 1560
acaggcacag ttaagttaca ttttatgaaa caatataata aatttaccga tatcgattat
1620 gcacatgcag atatgatgta aaaaagtttt tccgtacaat aatcattaag
atgataaaat 1680 tgtacggttt ttattttgtt ctgaacgggt tg 1712 14 466 PRT
Staphylococcus aureus 14 Met Asp Arg Met Tyr Glu Gln Asn Gln Met
Pro His Asn Asn Glu Ala 1 5 10 15 Glu Gln Ser Val Leu Gly Ser Ile
Ile Ile Asp Pro Glu Leu Ile Asn 20 25 30 Thr Thr Gln Glu Val Leu
Leu Pro Glu Ser Phe Tyr Arg Gly Ala His 35 40 45 Gln His Ile Phe
Arg Ala Met Met His Leu Asn Glu Asp Asn Lys Glu 50 55 60 Ile Asp
Val Val Thr Leu Met Asp Gln Leu Ser Thr Glu Gly Thr Leu 65 70 75 80
Asn Glu Ala Gly Gly Pro Gln Tyr Leu Ala Glu Leu Ser Thr Asn Val 85
90 95 Pro Thr Thr Arg Asn Val Gln Tyr Tyr Thr Asp Ile Val Ser Lys
His 100 105 110 Ala Leu Lys Arg Arg Leu Ile Gln Thr Ala Asp Ser Ile
Ala Asn Asp 115 120 125 Gly Tyr Asn Asp Glu Leu Glu Leu Asp Ala Ile
Leu Ser Asp Ala Glu 130 135 140 Arg Arg Ile Leu Glu Leu Ser Ser Ser
Arg Glu Ser Asp Gly Phe Lys 145 150 155 160 Asp Ile Arg Asp Val Leu
Gly Gln Val Tyr Glu Thr Ala Glu Glu Leu 165 170 175 Asp Gln Asn Ser
Gly Gln Thr Pro Gly Ile Pro Thr Gly Tyr Arg Asp 180 185 190 Leu Asp
Gln Met Thr Ala Gly Phe Asn Arg Asn Asp Leu Ile Ile Leu 195 200 205
Ala Ala Arg Pro Ser Val Gly Lys Thr Ala Phe Ala Leu Asn Ile Ala 210
215 220 Gln Lys Val Ala Thr His Glu Asp Met Tyr Thr Val Gly Ile Phe
Ser 225 230 235 240 Leu Glu Met Gly Ala Asp Gln Leu Ala Thr Arg Met
Ile Cys Ser Ser 245 250 255 Gly Asn Val Asp Ser Asn Arg Leu Arg Thr
Gly Thr Met Thr Glu Glu 260 265 270 Asp Trp Ser Arg Phe Thr Ile Ala
Val Gly Lys Leu Ser Arg Thr Lys 275 280 285 Ile Phe Ile Asp Asp Thr
Pro Gly Ile Arg Ile Asn Asp Leu Arg Ser 290 295 300 Lys Cys Arg Arg
Leu Lys Gln Glu His Gly Leu Asp Met Ile Val Ile 305 310 315 320 Asp
Tyr Leu Gln Leu Ile Gln Gly Ser Gly Ser Arg Ala Ser Asp Asn 325 330
335 Arg Gln Gln Glu Val Ser Glu Ile Ser Arg Thr Leu Lys Ala Leu Ala
340 345 350 Arg Glu Leu Lys Cys Pro Val Ile Ala Leu Ser Gln Leu Ser
Arg Gly 355 360 365 Val Glu Gln Arg Gln Asp Lys Arg Pro Met Met Ser
Asp Ile Arg Glu 370 375 380 Ser Gly Ser Ile Glu Gln Asp Ala Asp Ile
Val Ala Phe Leu Tyr Arg 385 390 395 400 Asp Asp Tyr Tyr Asn Arg Gly
Gly Asp Glu Asp Asp Asp Asp Asp Gly 405 410 415 Gly Phe Glu Pro Gln
Thr Asn Asp Glu Asn Gly Glu Ile Glu Ile Ile 420 425 430 Ile Ala Lys
Gln Arg Asn Gly Pro Thr Gly Thr Val Lys Leu His Phe 435 440 445 Met
Lys Gln Tyr Asn Lys Phe Thr Asp Ile Asp Tyr Ala His Ala Asp 450 455
460 Met Met 465 15 1170 DNA Staphylococcus aureus 15 gtggttccgt
attattagga ttggaaggta ctgtagttaa agcacacggt agttcaaatg 60
ctaaagcttt ttattctgca attagacaag cgaaaatcgc aggagaacaa aatattgtac
120 aaacaatgaa agagactgta ggtgaatcaa atgagtaaaa cagcaattat
ttttccggga 180 caaggtgccc aaaaagttgg tatggcgcaa gatttgttta
acaacaatga tcaagcaact 240 gaaattttaa cttcagcagc gaacacatta
gactttgata ttttagagac aatgtttact 300 gatgaagaag gtaaattggg
tgaaactgaa aacacacaac cagctttatt gacgcatagt 360 tcggcattat
tagcagcgct aaaaaatttg aatcctgatt ttactatggg gcatagttta 420
ggtgaatatt caagtttagt tgcagctgac gtattatcat ttgaagatgc agttaaaatt
480 gttagaaaac gtggtcaatt aatggcgcaa gcatttccta ctggtgtagg
aagcatggct 540 gcagtattgg gattagattt tgataaagtc gatgaaattt
gtaagtcatt atcatctgat 600 gacaaaataa ttgaaccagc aaacattaat
tgcccaggtc aaattgttgt ttcaggtcac 660 aaagctttaa ttgatgagct
agtagaaaaa ggtaaatcat taggtgcaaa acgtgtcatg 720 cctttagcag
tatctggacc attccattca tcgctaatga aagtgattga agaagatttt 780
tcaagttaca ttaatcaatt tgaatggcgt gatgctaagt ttcctgtagt tcaaaatgta
840 aatgcgcaag gtgaaactga caaagaagta attaaatcta atatggtcaa
gcaattatat 900 tcaccagtac aattcattaa ctcaacagaa tggctaatag
accaaggtgt tgatcatttt 960 attgaaattg gtcctggaaa agttttatct
ggcttaatta aaaaaataaa tagagatgtt 1020 aagttaacat caattcaaac
tttagaagat gtgaaaggat ggaatgaaaa tgactaagag 1080 tgctttagta
acaggtgcat caagaggaat tggacgtagt attgcgttac aattagcaga 1140
agaaggatat aatgtagcag taaactatgc 1170 16 308 PRT Staphylococcus
aureus 16 Met Ser Lys Thr Ala Ile Ile Phe Pro Gly Gln Gly Ala Gln
Lys Val 1 5 10 15 Gly Met Ala Gln Asp Leu Phe Asn Asn Asn Asp Gln
Ala Thr Glu Ile 20 25 30 Leu Thr Ser Ala Ala Asn Thr Leu Asp Phe
Asp Ile Leu Glu Thr Met 35 40 45 Phe Thr Asp Glu Glu Gly Lys Leu
Gly Glu Thr Glu Asn Thr Gln Pro 50 55 60 Ala Leu Leu Thr His Ser
Ser Ala Leu Leu Ala Ala Leu Lys Asn Leu 65 70 75 80 Asn Pro Asp Phe
Thr Met Gly His Ser Leu Gly Glu Tyr Ser Ser Leu 85 90 95 Val Ala
Ala Asp Val Leu Ser Phe Glu Asp Ala Val Lys Ile Val Arg 100 105 110
Lys Arg Gly Gln Leu Met Ala Gln Ala Phe Pro Thr Gly Val Gly Ser 115
120 125 Met Ala Ala Val Leu Gly Leu Asp Phe Asp Lys Val Asp Glu Ile
Cys 130 135 140 Lys Ser Leu Ser Ser Asp Asp Lys Ile Ile Glu Pro Ala
Asn Ile Asn 145 150 155 160 Cys Pro Gly Gln Ile Val Val Ser Gly His
Lys Ala Leu Ile Asp Glu 165 170 175 Leu Val Glu Lys Gly Lys Ser Leu
Gly Ala Lys Arg Val Met Pro Leu 180 185 190 Ala Val Ser Gly Pro Phe
His Ser Ser Leu Met Lys Val Ile Glu Glu 195 200 205 Asp Phe Ser Ser
Tyr Ile Asn Gln Phe Glu Trp Arg Asp Ala Lys Phe 210 215 220 Pro Val
Val Gln Asn Val Asn Ala Gln Gly Glu Thr Asp Lys Glu Val 225 230 235
240 Ile Lys Ser Asn Met Val Lys Gln Leu Tyr Ser Pro Val Gln Phe Ile
245 250 255 Asn Ser Thr Glu Trp Leu Ile Asp Gln Gly Val Asp His Phe
Ile Glu 260 265 270 Ile Gly Pro Gly Lys Val Leu Ser Gly Leu Ile Lys
Lys Ile Asn Arg 275 280 285 Asp Val Lys Leu Thr Ser Ile Gln Thr Leu
Glu Asp Val Lys Gly Trp 290 295 300 Asn Glu Asn Asp 305 17 1080 DNA
Staphylococcus aureus 17 aaatacacat ttaatctgca gtatttcaat
gcattgacgc tatttttttg atataattac 60 tttgaaaaat acgtgcgtaa
gcactcaagg aggaactttc atgcctttag tttcaatgaa 120 agaaatgtta
attgatgcaa aagaaaatgg ttatgcggta ggtcaataca atattaataa 180
cctagaattc actcaagcaa ttttagaagc gtcacaagaa gaaaatgcac ctgtaatttt
240 aggtgtttct gaaggtgctg ctcgttacat gagcggtttc tacacaattg
ttaaaatggt 300 tgaagggtta atgcatgact taaacatcac tattcctgta
gcaatccatt tagaccatgg 360 ttcaagcttt gaaaaatgta aagaagctat
cgatgctggt ttcacatcag taatgatcga 420 tgcttcacac agcccattcg
aagaaaacgt agcaacaact aaaaaagttg ttgaatacgc 480 tcatgaaaaa
ggtgtttctg tagaagctga attaggtact gttggtggac aagaagatga 540
tgttgtagca gacggcatca tttatgctga tcctaaagaa tgtcaagaac tagttgaaaa
600 aactggtatt gatgcattag cgccagcatt aggttcagtt catggtccat
acaaaggtga 660 accaaaatta ggatttaaag aaatggaaga aatcggttta
tctacaggtt taccattagt 720 attacacggt ggtactggta tcccgactaa
agatatccaa aaagcaattc catttggtac 780 agctaaaatt aacgtaaaca
ctgaaaacca aatcgcttca gcaaaagcag ttcgtgacgt 840 tttaaataac
gacaaagaag tttacgatcc tcgtaaatac ttaggacctg cacgtgaagc 900
catcaaagaa acagttaaag gtaaaattaa agagttcggt acttctaacc gcgctaaata
960 attaatattt agtctttaag ttattaataa cgtagggata ttaattttaa
aagaagcaga 1020 caaaatggtg tttgcttctt ttttatgtcg tataagtaat
aaataaaaca gtttgatttt 1080 18 286 PRT Staphylococcus aureus 18 Met
Pro Leu Val Ser Met Lys Glu Met Leu Ile Asp Ala Lys Glu Asn 1 5 10
15 Gly Tyr Ala Val Gly Gln Tyr Asn Ile Asn Asn Leu Glu Phe Thr Gln
20 25 30 Ala Ile Leu Glu Ala Ser Gln Glu Glu Asn Ala Pro Val Ile
Leu Gly 35 40 45 Val Ser Glu Gly Ala Ala Arg Tyr Met Ser Gly Phe
Tyr Thr Ile Val 50 55 60 Lys Met Val Glu Gly Leu Met His Asp Leu
Asn Ile Thr Ile Pro Val 65 70 75 80 Ala Ile His Leu Asp His Gly Ser
Ser Phe Glu Lys Cys Lys Glu Ala 85 90 95 Ile Asp Ala Gly Phe Thr
Ser Val Met Ile Asp Ala Ser His Ser Pro 100 105 110 Phe Glu Glu Asn
Val Ala Thr Thr Lys Lys Val Val Glu Tyr Ala His 115 120 125 Glu Lys
Gly Val Ser Val Glu Ala Glu Leu Gly Thr Val Gly Gly Gln 130 135 140
Glu Asp Asp Val Val Ala Asp Gly Ile Ile Tyr Ala Asp Pro Lys Glu 145
150 155 160 Cys Gln Glu Leu Val Glu Lys Thr Gly Ile Asp Ala Leu Ala
Pro Ala 165 170 175 Leu Gly Ser Val His Gly Pro Tyr Lys Gly Glu Pro
Lys Leu Gly Phe 180 185 190 Lys Glu Met Glu Glu Ile Gly Leu Ser Thr
Gly Leu Pro Leu Val Leu 195 200 205 His Gly Gly Thr Gly Ile Pro Thr
Lys Asp Ile Gln Lys Ala Ile Pro 210 215 220 Phe Gly Thr Ala Lys Ile
Asn Val Asn Thr Glu Asn Gln Ile Ala Ser 225 230 235 240 Ala Lys Ala
Val Arg Asp Val Leu Asn Asn Asp Lys Glu Val Tyr Asp 245 250 255 Pro
Arg Lys Tyr Leu Gly Pro Ala Arg Glu Ala Ile Lys Glu Thr Val 260 265
270 Lys Gly Lys Ile Lys Glu Phe Gly Thr Ser Asn Arg Ala Lys 275 280
285 19 1340 DNA Staphylococcus aureus 19 gctataatag gcatggttac
aatgagcttg ctcatacata ttaatataat tacaaaaaca 60 cgtcggaggt
acgacatgat taaaaataca attaaaaaat tgatagaaca tagtatatat 120
acgactttta aattactatc aaaattgcca aacaagaatc taatttattt tgaaagcttt
180 catggtaaac aatacagcga caaccccaaa gcattatatg aatacttaac
tgaacatagc 240 gatgcccaat taatatgggg tgtgaaaaaa ggatatgaac
acatattcca acagcacaat 300 gtaccatatg ttacaaagtt ttcaatgaaa
tggtttttag cgatgccaag agcgaaagcg 360 tggatgatta acacacgtac
accagattgg ttatataaat caccgcgaac gacgtactta 420 caaacatggc
atggcacgcc attaaaaaag attggtttgg atattagtaa cgttaaaatg 480
ctaggaacaa atactcaaaa ttaccaagat ggctttaaaa aagaaagcca acggtgggat
540 tatctagtgt cacctaatcc atattcgaca tcgatatttc aaaatgcatt
tcatgttagt 600 cgagataaga ttttggaaac aggttatcca agaaatgata
aattatcaca taaacgcaat 660 gatactgaat atattaatgg tattaagaca
agattaaata ttccattaga taaaaaagtg 720 attatgtacg cgccaacttg
gcgtgacgat gaagcgattc gagaaggttc atatcaattt 780 aatgttaact
ttgatataga agctttgcgt caagcgctgg atgatgatta tgttatttta 840
ttacgcatgc attatttagt tgtgacacgt attgatgaac atgatgattt tgtgaaagac
900 gtttcagatt atgaagacat ttcggattta tacttaatca
gcgatgcgtt agttaccgac 960 tactcatctg tcatgttcga cttcggtgta
ttaaagcgtc cgcaaatttt ctatgcatat 1020 gacttagata aatatggcga
tgagcttaga ggtttttaca tggattataa aaaagagttg 1080 ccaggtccaa
ttgttgaaaa tcaaacagca ctcattgatg cattaaaaca aatcgatgag 1140
actgcaaatg agtatattga agcacgaacg gtattttatc aaaaattctg ttcattagaa
1200 gatggacaag cgtcacaacg aatttgccaa acgattttta agtgataact
taaaaacaat 1260 aaaaaattat aaattaatta gttaagtgat ataaataata
aacgaaatgt ttgcttgtat 1320 gttattattt gtgtatgaaa 1340 20 389 PRT
Staphylococcus aureus 20 Met Ile Lys Asn Thr Ile Lys Lys Leu Ile
Glu His Ser Ile Tyr Thr 1 5 10 15 Thr Phe Lys Leu Leu Ser Lys Leu
Pro Asn Lys Asn Leu Ile Tyr Phe 20 25 30 Glu Ser Phe His Gly Lys
Gln Tyr Ser Asp Asn Pro Lys Ala Leu Tyr 35 40 45 Glu Tyr Leu Thr
Glu His Ser Asp Ala Gln Leu Ile Trp Gly Val Lys 50 55 60 Lys Gly
Tyr Glu His Ile Phe Gln Gln His Asn Val Pro Tyr Val Thr 65 70 75 80
Lys Phe Ser Met Lys Trp Phe Leu Ala Met Pro Arg Ala Lys Ala Trp 85
90 95 Met Ile Asn Thr Arg Thr Pro Asp Trp Leu Tyr Lys Ser Pro Arg
Thr 100 105 110 Thr Tyr Leu Gln Thr Trp His Gly Thr Pro Leu Lys Lys
Ile Gly Leu 115 120 125 Asp Ile Ser Asn Val Lys Met Leu Gly Thr Asn
Thr Gln Asn Tyr Gln 130 135 140 Asp Gly Phe Lys Lys Glu Ser Gln Arg
Trp Asp Tyr Leu Val Ser Pro 145 150 155 160 Asn Pro Tyr Ser Thr Ser
Ile Phe Gln Asn Ala Phe His Val Ser Arg 165 170 175 Asp Lys Ile Leu
Glu Thr Gly Tyr Pro Arg Asn Asp Lys Leu Ser His 180 185 190 Lys Arg
Asn Asp Thr Glu Tyr Ile Asn Gly Ile Lys Thr Arg Leu Asn 195 200 205
Ile Pro Leu Asp Lys Lys Val Ile Met Tyr Ala Pro Thr Trp Arg Asp 210
215 220 Asp Glu Ala Ile Arg Glu Gly Ser Tyr Gln Phe Asn Val Asn Phe
Asp 225 230 235 240 Ile Glu Ala Leu Arg Gln Ala Leu Asp Asp Asp Tyr
Val Ile Leu Leu 245 250 255 Arg Met His Tyr Leu Val Val Thr Arg Ile
Asp Glu His Asp Asp Phe 260 265 270 Val Lys Asp Val Ser Asp Tyr Glu
Asp Ile Ser Asp Leu Tyr Leu Ile 275 280 285 Ser Asp Ala Leu Val Thr
Asp Tyr Ser Ser Val Met Phe Asp Phe Gly 290 295 300 Val Leu Lys Arg
Pro Gln Ile Phe Tyr Ala Tyr Asp Leu Asp Lys Tyr 305 310 315 320 Gly
Asp Glu Leu Arg Gly Phe Tyr Met Asp Tyr Lys Lys Glu Leu Pro 325 330
335 Gly Pro Ile Val Glu Asn Gln Thr Ala Leu Ile Asp Ala Leu Lys Gln
340 345 350 Ile Asp Glu Thr Ala Asn Glu Tyr Ile Glu Ala Arg Thr Val
Phe Tyr 355 360 365 Gln Lys Phe Cys Ser Leu Glu Asp Gly Gln Ala Ser
Gln Arg Ile Cys 370 375 380 Gln Thr Ile Phe Lys 385 21 1430 DNA
Staphylococcus aureus 21 tgatttgtaa tcaaaactag atataattaa
ataatgactt aaaataattt taaaataggg 60 aaatgtaaag taataggagt
tctaagtgga ggatttacga tggataaaat agtaatcaaa 120 ggtggaaata
aattaacggg tgaagttaaa gtagaaggtg ctaaaaatgc agtattacca 180
atattgacag catctttatt agcttctgat aaaccgagca aattagttaa tgttccagct
240 ttaagtgatg tagaaacaat aaataatgta ttaacaactt taaatgctga
cgttacatac 300 aaaaaggacg aaaatgctgt tgtcgttgat gcaacaaaga
ctctaaatga agaggcacca 360 tatgaatatg ttagtaaaat gcgtgcaagt
attttagtta tgggacctct tttagcaaga 420 ctaggacatg ctattgttgc
attgcctggt ggttgtgcaa ttggaagtag accgattgag 480 caacacatta
aaggttttga agctttaggc gcagaaattc atcttgaaaa tggtaatatt 540
tatgctaatg ctaaagatgg attaaaaggt acatcaattc atttagattt tccaagtgta
600 ggagcaacac aaaatattat tatggcagca tcattagcta agggtaagac
tttaattgaa 660 aatgcagcta aagaacctga aattgtcgat ttagcaaact
acattaatga aatgggtggt 720 agaattactg gtgctggtac agacacaatt
acaatcaatg gtgtagaatc attacatggt 780 gtagaacatg ctatcattcc
agatagaatt gaagcaggca cattactaat cgctggtgct 840 ataacgcgtg
gtgatatttt tgtacgtggt gcaatcaaag aacatatggc gagtttagtc 900
tataaactag aagaaatggg cgttgaattg gactatcaag aagatggtat tcgtgtacgt
960 gctgaagggg aattacaacc tgtagacatc aaaactctac cacatcctgg
attcccgact 1020 gatatgcaat cacaaatgat ggcattgtta ttaacggcaa
atggtcataa agtcgtaacc 1080 gaaactgttt ttgaaaaccg ttttatgcat
gttgcagagt tcaaacgtat gaatgctaat 1140 atcaatgtag aaggtcgtag
tgctaaactt gaaggtaaaa gtcaattgca aggtgcacaa 1200 gttaaagcga
ctgatttaag agcagcagcc gccttaattt tagctggatt agttgctgat 1260
ggtaaaacaa gcgttactga attaacgcac ctagatagag gctatgttga cttacacggt
1320 aaattgaagc aattaggtgc agacattgaa cgtattaacg attaattcag
taaattaata 1380 taatggagga tttcaaccat ggaaacaatt tttgattata
accaaattaa 1430 22 421 PRT Staphylococcus aureus 22 Met Asp Lys Ile
Val Ile Lys Gly Gly Asn Lys Leu Thr Gly Glu Val 1 5 10 15 Lys Val
Glu Gly Ala Lys Asn Ala Val Leu Pro Ile Leu Thr Ala Ser 20 25 30
Leu Leu Ala Ser Asp Lys Pro Ser Lys Leu Val Asn Val Pro Ala Leu 35
40 45 Ser Asp Val Glu Thr Ile Asn Asn Val Leu Thr Thr Leu Asn Ala
Asp 50 55 60 Val Thr Tyr Lys Lys Asp Glu Asn Ala Val Val Val Asp
Ala Thr Lys 65 70 75 80 Thr Leu Asn Glu Glu Ala Pro Tyr Glu Tyr Val
Ser Lys Met Arg Ala 85 90 95 Ser Ile Leu Val Met Gly Pro Leu Leu
Ala Arg Leu Gly His Ala Ile 100 105 110 Val Ala Leu Pro Gly Gly Cys
Ala Ile Gly Ser Arg Pro Ile Glu Gln 115 120 125 His Ile Lys Gly Phe
Glu Ala Leu Gly Ala Glu Ile His Leu Glu Asn 130 135 140 Gly Asn Ile
Tyr Ala Asn Ala Lys Asp Gly Leu Lys Gly Thr Ser Ile 145 150 155 160
His Leu Asp Phe Pro Ser Val Gly Ala Thr Gln Asn Ile Ile Met Ala 165
170 175 Ala Ser Leu Ala Lys Gly Lys Thr Leu Ile Glu Asn Ala Ala Lys
Glu 180 185 190 Pro Glu Ile Val Asp Leu Ala Asn Tyr Ile Asn Glu Met
Gly Gly Arg 195 200 205 Ile Thr Gly Ala Gly Thr Asp Thr Ile Thr Ile
Asn Gly Val Glu Ser 210 215 220 Leu His Gly Val Glu His Ala Ile Ile
Pro Asp Arg Ile Glu Ala Gly 225 230 235 240 Thr Leu Leu Ile Ala Gly
Ala Ile Thr Arg Gly Asp Ile Phe Val Arg 245 250 255 Gly Ala Ile Lys
Glu His Met Ala Ser Leu Val Tyr Lys Leu Glu Glu 260 265 270 Met Gly
Val Glu Leu Asp Tyr Gln Glu Asp Gly Ile Arg Val Arg Ala 275 280 285
Glu Gly Glu Leu Gln Pro Val Asp Ile Lys Thr Leu Pro His Pro Gly 290
295 300 Phe Pro Thr Asp Met Gln Ser Gln Met Met Ala Leu Leu Leu Thr
Ala 305 310 315 320 Asn Gly His Lys Val Val Thr Glu Thr Val Phe Glu
Asn Arg Phe Met 325 330 335 His Val Ala Glu Phe Lys Arg Met Asn Ala
Asn Ile Asn Val Glu Gly 340 345 350 Arg Ser Ala Lys Leu Glu Gly Lys
Ser Gln Leu Gln Gly Ala Gln Val 355 360 365 Lys Ala Thr Asp Leu Arg
Ala Ala Ala Ala Leu Ile Leu Ala Gly Leu 370 375 380 Val Ala Asp Gly
Lys Thr Ser Val Thr Glu Leu Thr His Leu Asp Arg 385 390 395 400 Gly
Tyr Val Asp Leu His Gly Lys Leu Lys Gln Leu Gly Ala Asp Ile 405 410
415 Glu Arg Ile Asn Asp 420 23 2204 DNA Staphylococcus aureus 23
agaaaaatgg ctcaatcgaa ctagatatta tctttaaatc acaagggcca aaacgtttgt
60 tagcgcaatt tgcaccaatt gaaaaaagga ggattaaggg atggctgatt
tatcgtctcg 120 tgtgaacgag ttacatgatt tattaaatca atacagttat
gaatactatg tagaggataa 180 tccatctgta ccagatagtg aatatgacaa
attacttcat gaactgatta aaatagaaga 240 ggagcatcct gagtataaga
ctgtagattc tccaacagtt agagttggcg gtgaagccca 300 agcctctttc
aataaagtca accatgacac gccaatgtta agtttaggga atgcatttaa 360
tgaggatgat ttgagaaaat tcgaccaacg catacgtgaa caaattggca acgttgaata
420 tatgtgcgaa ttaaaaattg atggcttagc agtatcattg aaatatgttg
atggatactt 480 cgttcaaggt ttaacacgtg gtgatggaac aacaggtgaa
gatattaccg aaaatttaaa 540 aacaattcat gcgatacctt tgaaaatgaa
agaaccatta aatgtagaag ttcgtggtga 600 agcatatatg ccgagacgtt
catttttacg attaaatgaa gaaaaagaaa aaaatgatga 660 gcagttattt
gcaaatccaa gaaacgctgc tgcgggatca ttaagacagt tagattctaa 720
attaacggca aaacgaaagc taagcgtatt tatatatagt gtcaatgatt tcactgattt
780 caatgcgcgt tcgcaaagtg aagcattaga tgagttagat aaattaggtt
ttacaacgaa 840 taaaaataga gcgcgtgtaa ataatatcga tggtgtttta
gagtatattg aaaaatggac 900 aagccaaaga gagtcattac cttatgatat
tgatgggatt gttattaagg ttaatgattt 960 agatcaacag gatgagatgg
gattcacaca aaaatctcct agatgggcca ttgcttataa 1020 atttccagct
gaggaagtag taactaaatt attagatatt gaattaagta ttggacgaac 1080
aggtgtagtc acacctactg ctattttaga accagtaaaa gtrgctggta caactgtatc
1140 aagagcatct ttgcacaatg aggatttaat tcatgacaga gatattcgaa
ttggtgatag 1200 tgttgtagtg aaaaaagcag gtgacatcat acctgaagtt
gtacgtagta ttccagaacg 1260 tagacctgag gatgctgtca catatcatat
gccaacccat tgtccaagtt gtggacatga 1320 attagtacgt attgaaggcg
aagtagcact tcgttgcatt aatccaaaat gccaagcaca 1380 acttgttgaa
ggattgattc actttgtatc aagacaagcc atgaatattg atggtttagg 1440
cactaaaatt attcaacagc tttatcaaag cgaattaatt aaagatgttg ctgatatttt
1500 ctatttaaca gaagaagatt tattaccttt agacagaatg gggcagaaaa
aagttgataa 1560 tttattagct gccattcaac aagctaagga caactcttta
gaaaatttat tatttggtct 1620 aggtattagg catttaggtg ttaaagcgag
ccaagtgtta gcagaaaaat atgaaacgat 1680 agatcgatta ctaacggtaa
ctgaagcgga attagtagaa attcatgata taggtgataa 1740 agtagcacaa
tctgtagtta cttatttaga aaatgaagat attcgtgctt taattcaaaa 1800
attaaaagat aaacatgtta atatgattta taaaggtatc aaaacatcag atattgaagg
1860 acatcctgaa tttagtggta aaacgatagt actgactggt aagytacatc
aaatgacacg 1920 caatgaagca tctaaatggc ttgcatcaca aggtgctaaa
gttacaagta gcgttactaa 1980 aaatacagat gtcgttattg ctggtgaaga
tgcaggttca aaattaacaa aagcacaaag 2040 tttaggtatt gaaatttgga
cagagcaaca atttgtagat aagcaaaatg aattaaatag 2100 ttagaggggt
atgtcgatga agcgtacatt agtattattg attacagcta tctttatact 2160
cgctgcttgt ggtaaccata aggatgacca ggctggaaaa gata 2204 24 667 PRT
Staphylococcus aureus 24 Met Ala Asp Leu Ser Ser Arg Val Asn Glu
Leu His Asp Leu Leu Asn 1 5 10 15 Gln Tyr Ser Tyr Glu Tyr Tyr Val
Glu Asp Asn Pro Ser Val Pro Asp 20 25 30 Ser Glu Tyr Asp Lys Leu
Leu His Glu Leu Ile Lys Ile Glu Glu Glu 35 40 45 His Pro Glu Tyr
Lys Thr Val Asp Ser Pro Thr Val Arg Val Gly Gly 50 55 60 Glu Ala
Gln Ala Ser Phe Asn Lys Val Asn His Asp Thr Pro Met Leu 65 70 75 80
Ser Leu Gly Asn Ala Phe Asn Glu Asp Asp Leu Arg Lys Phe Asp Gln 85
90 95 Arg Ile Arg Glu Gln Ile Gly Asn Val Glu Tyr Met Cys Glu Leu
Lys 100 105 110 Ile Asp Gly Leu Ala Val Ser Leu Lys Tyr Val Asp Gly
Tyr Phe Val 115 120 125 Gln Gly Leu Thr Arg Gly Asp Gly Thr Thr Gly
Glu Asp Ile Thr Glu 130 135 140 Asn Leu Lys Thr Ile His Ala Ile Pro
Leu Lys Met Lys Glu Pro Leu 145 150 155 160 Asn Val Glu Val Arg Gly
Glu Ala Tyr Met Pro Arg Arg Ser Phe Leu 165 170 175 Arg Leu Asn Glu
Glu Lys Glu Lys Asn Asp Glu Gln Leu Phe Ala Asn 180 185 190 Pro Arg
Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp Ser Lys Leu 195 200 205
Thr Ala Lys Arg Lys Leu Ser Val Phe Ile Tyr Ser Val Asn Asp Phe 210
215 220 Thr Asp Phe Asn Ala Arg Ser Gln Ser Glu Ala Leu Asp Glu Leu
Asp 225 230 235 240 Lys Leu Gly Phe Thr Thr Asn Lys Asn Arg Ala Arg
Val Asn Asn Ile 245 250 255 Asp Gly Val Leu Glu Tyr Ile Glu Lys Trp
Thr Ser Gln Arg Glu Ser 260 265 270 Leu Pro Tyr Asp Ile Asp Gly Ile
Val Ile Lys Val Asn Asp Leu Asp 275 280 285 Gln Gln Asp Glu Met Gly
Phe Thr Gln Lys Ser Pro Arg Trp Ala Ile 290 295 300 Ala Tyr Lys Phe
Pro Ala Glu Glu Val Val Thr Lys Leu Leu Asp Ile 305 310 315 320 Glu
Leu Ser Ile Gly Arg Thr Gly Val Val Thr Pro Thr Ala Ile Leu 325 330
335 Glu Pro Val Lys Val Ala Gly Thr Thr Val Ser Arg Ala Ser Leu His
340 345 350 Asn Glu Asp Leu Ile His Asp Arg Asp Ile Arg Ile Gly Asp
Ser Val 355 360 365 Val Val Lys Lys Ala Gly Asp Ile Ile Pro Glu Val
Val Arg Ser Ile 370 375 380 Pro Glu Arg Arg Pro Glu Asp Ala Val Thr
Tyr His Met Pro Thr His 385 390 395 400 Cys Pro Ser Cys Gly His Glu
Leu Val Arg Ile Glu Gly Glu Val Ala 405 410 415 Leu Arg Cys Ile Asn
Pro Lys Cys Gln Ala Gln Leu Val Glu Gly Leu 420 425 430 Ile His Phe
Val Ser Arg Gln Ala Met Asn Ile Asp Gly Leu Gly Thr 435 440 445 Lys
Ile Ile Gln Gln Leu Tyr Gln Ser Glu Leu Ile Lys Asp Val Ala 450 455
460 Asp Ile Phe Tyr Leu Thr Glu Glu Asp Leu Leu Pro Leu Asp Arg Met
465 470 475 480 Gly Gln Lys Lys Val Asp Asn Leu Leu Ala Ala Ile Gln
Gln Ala Lys 485 490 495 Asp Asn Ser Leu Glu Asn Leu Leu Phe Gly Leu
Gly Ile Arg His Leu 500 505 510 Gly Val Lys Ala Ser Gln Val Leu Ala
Glu Lys Tyr Glu Thr Ile Asp 515 520 525 Arg Leu Leu Thr Val Thr Glu
Ala Glu Leu Val Glu Ile His Asp Ile 530 535 540 Gly Asp Lys Val Ala
Gln Ser Val Val Thr Tyr Leu Glu Asn Glu Asp 545 550 555 560 Ile Arg
Ala Leu Ile Gln Lys Leu Lys Asp Lys His Val Asn Met Ile 565 570 575
Tyr Lys Gly Ile Lys Thr Ser Asp Ile Glu Gly His Pro Glu Phe Ser 580
585 590 Gly Lys Thr Ile Val Leu Thr Gly Lys Leu His Gln Met Thr Arg
Asn 595 600 605 Glu Ala Ser Lys Trp Leu Ala Ser Gln Gly Ala Lys Val
Thr Ser Ser 610 615 620 Val Thr Lys Asn Thr Asp Val Val Ile Ala Gly
Glu Asp Ala Gly Ser 625 630 635 640 Lys Leu Thr Lys Ala Gln Ser Leu
Gly Ile Glu Ile Trp Thr Glu Gln 645 650 655 Gln Phe Val Asp Lys Gln
Asn Glu Leu Asn Ser 660 665 25 959 DNA Staphylococcus aureus 25
tgtctcactc actttccaaa atactaaagt aacatcttta gtatatcaaa gaatttttgc
60 tataataagt tataattata taaaaaagga acgggataaa atgattgtaa
aaacagaaga 120 agaattacaa gcgttaaaag aaattggata catatgcgct
aaagtgcgca atacaatgca 180 agctgcaacc aaaccaggta tcactacgaa
agagcttgat aatattgcga aagagttatt 240 tgaagaatac ggtgctattt
ctgcgccaat tcatgatgaa aattttcctg gtcaaacgtg 300 tattagtgtc
aatgaagagg tggcacatgg gattccaagt aagcgtgtca ttcgtgaagg 360
agatttagta aatattgatg tatcggcttt gaagaatggc tattatgcag atacaggcat
420 ttcatttgtc gttggagaat cagatgatcc aatgaaacaa aaagtatgtg
acgtagcaac 480 gatggcattt gagaatgcaa ttgcaaaagt aaaaccgggt
actaagttaa gtaacattgg 540 taaagcggtg cataatacag ctagacaaaa
tgatttgaaa gtcattaaaa acttaacagg 600 tcatggtgtt ggtttatcat
tacatgaagc accagcacat gtacttaatt actttgatcc 660 aaaagacaaa
acattattaa ctgaaggtat ggtattagct attgaaccgt ttatctcatc 720
aaatgcatca tttgttacag aaggtaaaaa tgaatgggct tttgaaacga gcgataaaag
780 ttttgttgct caaattgagc atacggttat cgtgactaag gatggtccga
ttttaacgac 840 aaagattgaa gaagaatagt tcaacatata ctaagactaa
agtatgaaca tcatttagtt 900 ccggagccta ttcatattgg tttcggaact
gttttataat aattaagaac acaatcaat 959 26 252 PRT Staphylococcus
aureus 26 Met Ile Val Lys Thr Glu Glu Glu Leu Gln Ala Leu Lys Glu
Ile Gly 1 5 10 15 Tyr Ile Cys Ala Lys Val Arg Asn Thr Met Gln Ala
Ala Thr Lys Pro 20 25 30 Gly Ile Thr Thr Lys Glu Leu Asp Asn Ile
Ala Lys Glu Leu Phe Glu 35 40 45 Glu Tyr Gly Ala Ile Ser Ala Pro
Ile His Asp Glu Asn Phe Pro Gly 50 55 60 Gln Thr Cys Ile Ser Val
Asn Glu Glu Val Ala His Gly Ile Pro Ser 65 70 75 80 Lys Arg Val Ile
Arg Glu Gly Asp Leu Val Asn Ile Asp Val Ser Ala 85 90 95 Leu Lys
Asn Gly Tyr Tyr Ala Asp Thr Gly Ile Ser Phe Val Val Gly 100 105 110
Glu Ser Asp Asp Pro Met Lys Gln Lys Val Cys Asp
Val Ala Thr Met 115 120 125 Ala Phe Glu Asn Ala Ile Ala Lys Val Lys
Pro Gly Thr Lys Leu Ser 130 135 140 Asn Ile Gly Lys Ala Val His Asn
Thr Ala Arg Gln Asn Asp Leu Lys 145 150 155 160 Val Ile Lys Asn Leu
Thr Gly His Gly Val Gly Leu Ser Leu His Glu 165 170 175 Ala Pro Ala
His Val Leu Asn Tyr Phe Asp Pro Lys Asp Lys Thr Leu 180 185 190 Leu
Thr Glu Gly Met Val Leu Ala Ile Glu Pro Phe Ile Ser Ser Asn 195 200
205 Ala Ser Phe Val Thr Glu Gly Lys Asn Glu Trp Ala Phe Glu Thr Ser
210 215 220 Asp Lys Ser Phe Val Ala Gln Ile Glu His Thr Val Ile Val
Thr Lys 225 230 235 240 Asp Gly Pro Ile Leu Thr Thr Lys Ile Glu Glu
Glu 245 250 27 3400 DNA Staphylococcus aureus 27 tatacagttt
atatgaaatt aaagtagcac ctcataaata cttagatttt taattggaaa 60
tttgatacaa tttagtgatg aatgacttaa aggaggcttt tattaatgac aaaagtaaca
120 cgtgaagaag ttgagcatat cgcgaatctt gcaagacttc aaatttctcc
tgaagaaacg 180 gaagaaatgg ccaacacatt agaaagcatt ttagattttg
caaaacaaaa tgatagcgct 240 gatacagaag gcgttgaacc tacatatcac
gttttagatt tacaaaacgt tttacgtgaa 300 gataaagcaa ttaaaggtat
tccacaagaa ttagctttga aaaatgccaa agaaacagaa 360 gatggacaat
ttaaagtgcc tacaatcatg aatgaggagg acgcgtaaga tgagcattcg 420
ctacgaatcg gttgagaatt tattaacttt aataaaagac aaaaaaatca aaccatctga
480 tgttgttaaa gatatatatg atgcaattga agagactgat ccaacaatta
agtcttttct 540 agcgctggat aaagaaaatg caatcaaaaa agcgcaagaa
ttggatgaat tacaagcaaa 600 agatcaaatg gatggcaaat tatttggtat
tccaatgggt ataaaagata acattattac 660 aaacggatta gaaacaacat
gtgcaagtaa aatgttagaa ggttttgtgc caatttacga 720 atctactgta
atggaaaaac tacataatga aaatgccgtt ttaatcggta aattaaatat 780
ggatgagttt gcaatgggtg gttcaacaga aacatcttat ttcaaaaaaa cagttaaccc
840 atttgaccat aaagcagtgc caggtggttc atcaggtgga tctgcagcag
cagttgcagc 900 tggcttagta ccatttagct taggttcaga cacaggtggt
tcaattagac aaccggctgc 960 atattgtggc gttgtcggta tgaaaccaac
atacggtcgt gtatctcgat ttggattagt 1020 tgcttttgca tcttcattag
accaaattgg tccattgact cgaaatgtaa aagataatgc 1080 aatcgtatta
gaagctattt ctggtgcaga tgttaatgac tctacaagtg caccagttga 1140
tgatgtagac tttacatctg aaattggtaa agatattaaa ggattaaaag ttgcattacc
1200 taaagaatac ttaggtgaag gtgtagctga tgacgtaaaa gaagcagttc
aaaacgctgt 1260 agaaacttta aaatctttag gtgctgtcgt tgaggaagta
tcattgccaa atactaaatt 1320 tggtattcca tcatattacg tgattgcatc
atcagaagct tcgtcaaacc tttctcgttt 1380 tgacggaatt cgttatggtt
atcattctaa agaagctcat tcattagaag aattatataa 1440 aatgtcaaga
tctgaaggtt tcggtaaaga agtaaaacgt cgtattttct taggtacatt 1500
tgcattaagt tcaggttact atgatgctta ctataaaaaa tctcaaaaag ttagaacatt
1560 gattaaaaat gactttgata aagtattcga aaattatgat gtagtagttg
gtccaacagc 1620 gcctacaact gcgtttaatt taggtgaaga aattgatgat
ccattaacaa tgtatgccaa 1680 tgatttatta acaacaccag taaacttagc
tggattacct ggtatttctg ttccttgtgg 1740 acaatcaaat ggccgaccaa
tcggtttaca gttcattggt aaaccattcg atgaaaaaac 1800 gttatatcgt
gtcgcttatc aatatgaaac acaatacaat ttacatgacg tttatgaaaa 1860
attataagga gtggaaatca tgcattttga aacagttata ggacttgaag ttcacgtaga
1920 gttaaaaacg gactcaaaaa tgttttctcc atcaccagcg cattttggag
cagaacctaa 1980 ctcaaataca aatgttatcg acttagcata tccaggtgtc
ttaccagttg ttaataagcg 2040 tgcagtagac tgggcaatgc gtgctgcaat
ggcactaaat atggaaatcg caacagaatc 2100 taagtttgac cgtaagaact
atttctatcc agataatcca aaagcatatc aaatttctca 2160 atttgatcaa
ccaattggtg aaaatggata tatcgatatc gaagtcgacg gtgaaacaaa 2220
acgaatcggt attactcgtc ttcacatgga agaagatgct ggtaagtcaa cacataaagg
2280 tgagtattca ttagttgact tgaaccgtca aggtacaccg ctaattgaaa
tcgtatctga 2340 accagatatt cgttcaccta aagaagcata tgcatattta
gaaaaattgc gttcaattat 2400 tcaatacact ggtgtatcag acgttaagat
ggaagaggga tctttacgtt gtgatgctaa 2460 catctcttta cgtccatatg
gtcaagaaaa atttggtact aaagccgaat tgaaaaactt 2520 aaactcattt
aactatgtac gtaaaggttt agaatatgaa gaaaaacgcc aagaagaaga 2580
attgttaaat ggtggagaaa tcggacaaga aacacgtcga tttgatgaat ctacaggtaa
2640 aacaatttta atgcgtgtta aagaaggttc tgatgattac cgttacttcc
cagagcctga 2700 cattgtacct ttatatattg atgatgcttg gaaagagcgt
gttcgtcaga caattcctga 2760 attaccagat gaacgtaaag ctaagtatgt
aaatgaatta ggtttacctg catacgatgc 2820 acacgtatta acattgacta
aagaaatgtc agatttcttt gaatcaacaa ttgaacacgg 2880 tgcagatgtt
aaattaacat ctaactggtt aatgggtggc gtaaacgaat atttaaataa 2940
aaatcaagta gaattattag atactaaatt aacaccagaa aatttagcag gtatgattaa
3000 acttatcgaa gacggaacaa tgagcagtaa aattgcgaag aaagtcttcc
cagagttagc 3060 agctaaaggt ggtaatgcta aacagattat ggaagataat
ggcttagttc aaatttctga 3120 tgaagcaaca cttctaaaat ttgtaaatga
agcattagac aataacgaac aatcagttga 3180 agattacaaa aatggtaaag
gcaaagctat gggcttctta gttggtcaaa ttatgaaagc 3240 gtctaaaggt
caagctaatc cacaattagt aaatcaacta ttaaaacaag aattagataa 3300
aagataattt aaatcatcaa actatgaaga tttaaaaaat aaacccttga ttgctgactt
3360 agatgcaatc gagggtttat ttatatctat agaagtcaaa 3400 28 485 PRT
Staphylococcus aureus 28 Met Ser Ile Arg Tyr Glu Ser Val Glu Asn
Leu Leu Thr Leu Ile Lys 1 5 10 15 Asp Lys Lys Ile Lys Pro Ser Asp
Val Val Lys Asp Ile Tyr Asp Ala 20 25 30 Ile Glu Glu Thr Asp Pro
Thr Ile Lys Ser Phe Leu Ala Leu Asp Lys 35 40 45 Glu Asn Ala Ile
Lys Lys Ala Gln Glu Leu Asp Glu Leu Gln Ala Lys 50 55 60 Asp Gln
Met Asp Gly Lys Leu Phe Gly Ile Pro Met Gly Ile Lys Asp 65 70 75 80
Asn Ile Ile Thr Asn Gly Leu Glu Thr Thr Cys Ala Ser Lys Met Leu 85
90 95 Glu Gly Phe Val Pro Ile Tyr Glu Ser Thr Val Met Glu Lys Leu
His 100 105 110 Asn Glu Asn Ala Val Leu Ile Gly Lys Leu Asn Met Asp
Glu Phe Ala 115 120 125 Met Gly Gly Ser Thr Glu Thr Ser Tyr Phe Lys
Lys Thr Val Asn Pro 130 135 140 Phe Asp His Lys Ala Val Pro Gly Gly
Ser Ser Gly Gly Ser Ala Ala 145 150 155 160 Ala Val Ala Ala Gly Leu
Val Pro Phe Ser Leu Gly Ser Asp Thr Gly 165 170 175 Gly Ser Ile Arg
Gln Pro Ala Ala Tyr Cys Gly Val Val Gly Met Lys 180 185 190 Pro Thr
Tyr Gly Arg Val Ser Arg Phe Gly Leu Val Ala Phe Ala Ser 195 200 205
Ser Leu Asp Gln Ile Gly Pro Leu Thr Arg Asn Val Lys Asp Asn Ala 210
215 220 Ile Val Leu Glu Ala Ile Ser Gly Ala Asp Val Asn Asp Ser Thr
Ser 225 230 235 240 Ala Pro Val Asp Asp Val Asp Phe Thr Ser Glu Ile
Gly Lys Asp Ile 245 250 255 Lys Gly Leu Lys Val Ala Leu Pro Lys Glu
Tyr Leu Gly Glu Gly Val 260 265 270 Ala Asp Asp Val Lys Glu Ala Val
Gln Asn Ala Val Glu Thr Leu Lys 275 280 285 Ser Leu Gly Ala Val Val
Glu Glu Val Ser Leu Pro Asn Thr Lys Phe 290 295 300 Gly Ile Pro Ser
Tyr Tyr Val Ile Ala Ser Ser Glu Ala Ser Ser Asn 305 310 315 320 Leu
Ser Arg Phe Asp Gly Ile Arg Tyr Gly Tyr His Ser Lys Glu Ala 325 330
335 His Ser Leu Glu Glu Leu Tyr Lys Met Ser Arg Ser Glu Gly Phe Gly
340 345 350 Lys Glu Val Lys Arg Arg Ile Phe Leu Gly Thr Phe Ala Leu
Ser Ser 355 360 365 Gly Tyr Tyr Asp Ala Tyr Tyr Lys Lys Ser Gln Lys
Val Arg Thr Leu 370 375 380 Ile Lys Asn Asp Phe Asp Lys Val Phe Glu
Asn Tyr Asp Val Val Val 385 390 395 400 Gly Pro Thr Ala Pro Thr Thr
Ala Phe Asn Leu Gly Glu Glu Ile Asp 405 410 415 Asp Pro Leu Thr Met
Tyr Ala Asn Asp Leu Leu Thr Thr Pro Val Asn 420 425 430 Leu Ala Gly
Leu Pro Gly Ile Ser Val Pro Cys Gly Gln Ser Asn Gly 435 440 445 Arg
Pro Ile Gly Leu Gln Phe Ile Gly Lys Pro Phe Asp Glu Lys Thr 450 455
460 Leu Tyr Arg Val Ala Tyr Gln Tyr Glu Thr Gln Tyr Asn Leu His Asp
465 470 475 480 Val Tyr Glu Lys Leu 485 29 475 PRT Staphylococcus
aureus 29 Met His Phe Glu Thr Val Ile Gly Leu Glu Val His Val Glu
Leu Lys 1 5 10 15 Thr Asp Ser Lys Met Phe Ser Pro Ser Pro Ala His
Phe Gly Ala Glu 20 25 30 Pro Asn Ser Asn Thr Asn Val Ile Asp Leu
Ala Tyr Pro Gly Val Leu 35 40 45 Pro Val Val Asn Lys Arg Ala Val
Asp Trp Ala Met Arg Ala Ala Met 50 55 60 Ala Leu Asn Met Glu Ile
Ala Thr Glu Ser Lys Phe Asp Arg Lys Asn 65 70 75 80 Tyr Phe Tyr Pro
Asp Asn Pro Lys Ala Tyr Gln Ile Ser Gln Phe Asp 85 90 95 Gln Pro
Ile Gly Glu Asn Gly Tyr Ile Asp Ile Glu Val Asp Gly Glu 100 105 110
Thr Lys Arg Ile Gly Ile Thr Arg Leu His Met Glu Glu Asp Ala Gly 115
120 125 Lys Ser Thr His Lys Gly Glu Tyr Ser Leu Val Asp Leu Asn Arg
Gln 130 135 140 Gly Thr Pro Leu Ile Glu Ile Val Ser Glu Pro Asp Ile
Arg Ser Pro 145 150 155 160 Lys Glu Ala Tyr Ala Tyr Leu Glu Lys Leu
Arg Ser Ile Ile Gln Tyr 165 170 175 Thr Gly Val Ser Asp Val Lys Met
Glu Glu Gly Ser Leu Arg Cys Asp 180 185 190 Ala Asn Ile Ser Leu Arg
Pro Tyr Gly Gln Glu Lys Phe Gly Thr Lys 195 200 205 Ala Glu Leu Lys
Asn Leu Asn Ser Phe Asn Tyr Val Arg Lys Gly Leu 210 215 220 Glu Tyr
Glu Glu Lys Arg Gln Glu Glu Glu Leu Leu Asn Gly Gly Glu 225 230 235
240 Ile Gly Gln Glu Thr Arg Arg Phe Asp Glu Ser Thr Gly Lys Thr Ile
245 250 255 Leu Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe
Pro Glu 260 265 270 Pro Asp Ile Val Pro Leu Tyr Ile Asp Asp Ala Trp
Lys Glu Arg Val 275 280 285 Arg Gln Thr Ile Pro Glu Leu Pro Asp Glu
Arg Lys Ala Lys Tyr Val 290 295 300 Asn Glu Leu Gly Leu Pro Ala Tyr
Asp Ala His Val Leu Thr Leu Thr 305 310 315 320 Lys Glu Met Ser Asp
Phe Phe Glu Ser Thr Ile Glu His Gly Ala Asp 325 330 335 Val Lys Leu
Thr Ser Asn Trp Leu Met Gly Gly Val Asn Glu Tyr Leu 340 345 350 Asn
Lys Asn Gln Val Glu Leu Leu Asp Thr Lys Leu Thr Pro Glu Asn 355 360
365 Leu Ala Gly Met Ile Lys Leu Ile Glu Asp Gly Thr Met Ser Ser Lys
370 375 380 Ile Ala Lys Lys Val Phe Pro Glu Leu Ala Ala Lys Gly Gly
Asn Ala 385 390 395 400 Lys Gln Ile Met Glu Asp Asn Gly Leu Val Gln
Ile Ser Asp Glu Ala 405 410 415 Thr Leu Leu Lys Phe Val Asn Glu Ala
Leu Asp Asn Asn Glu Gln Ser 420 425 430 Val Glu Asp Tyr Lys Asn Gly
Lys Gly Lys Ala Met Gly Phe Leu Val 435 440 445 Gly Gln Ile Met Lys
Ala Ser Lys Gly Gln Ala Asn Pro Gln Leu Val 450 455 460 Asn Gln Leu
Leu Lys Gln Glu Leu Asp Lys Arg 465 470 475 30 100 PRT
Staphylococcus aureus 30 Met Thr Lys Val Thr Arg Glu Glu Val Glu
His Ile Ala Asn Leu Ala 1 5 10 15 Arg Leu Gln Ile Ser Pro Glu Glu
Thr Glu Glu Met Ala Asn Thr Leu 20 25 30 Glu Ser Ile Leu Asp Phe
Ala Lys Gln Asn Asp Ser Ala Asp Thr Glu 35 40 45 Gly Val Glu Pro
Thr Tyr His Val Leu Asp Leu Gln Asn Val Leu Arg 50 55 60 Glu Asp
Lys Ala Ile Lys Gly Ile Pro Gln Glu Leu Ala Leu Lys Asn 65 70 75 80
Ala Lys Glu Thr Glu Asp Gly Gln Phe Lys Val Pro Thr Ile Met Asn 85
90 95 Glu Glu Asp Ala 100 31 772 DNA Staphylococcus aureus 31
cttactaagc taaagaataa tgataattga tggcaatggc ggaaaatgga tgttgtcatt
60 ataataataa atgaaacaat tatgttggag gtaaacacgc atgaaatgta
ttgtaggtct 120 aggtaatata ggtaaacgtt ttgaacttac aagacataat
atcggctttg aagtcgttga 180 ttatatttta gagaaaaata atttttcatt
agataaacaa aagtttaaag gtgcatatac 240 aattgaacga atgaacggcg
ataaagtgtt atttatcgaa ccaatgacaa tgatgaattt 300 gtcaggtgaa
gcagttgcac cgattatgga ttattacaat gttaatccag aagatttaat 360
tgtcttatat gatgatttag atttagaaca aggacaagtt cgcttaagac aaaaaggaag
420 tgcgggcggt cacaatggta tgaaatcaat tattaaaatg cttggtacag
accaatttaa 480 acgtattcgt attggtgtgg gaagaccaac gaatggtatg
acggtacctg attatgtttt 540 acaacgcttt tcaaatgatg aaatggtaac
gatggaaaaa gttatcgaac acgcagcacg 600 cgcaattgaa aagtttgttg
aaacatcacg atttgaccat gttatgaatg aatttaatgg 660 tgaagtgaaa
taatgacaat attgacaacg cttataaaag aagataatca ttttcaagac 720
cttaatcagg tatttggaca agcaaacaca ctagtaactg gtctttcccc gt 772 32
190 PRT Staphylococcus aureus 32 Met Lys Cys Ile Val Gly Leu Gly
Asn Ile Gly Lys Arg Phe Glu Leu 1 5 10 15 Thr Arg His Asn Ile Gly
Phe Glu Val Val Asp Tyr Ile Leu Glu Lys 20 25 30 Asn Asn Phe Ser
Leu Asp Lys Gln Lys Phe Lys Gly Ala Tyr Thr Ile 35 40 45 Glu Arg
Met Asn Gly Asp Lys Val Leu Phe Ile Glu Pro Met Thr Met 50 55 60
Met Asn Leu Ser Gly Glu Ala Val Ala Pro Ile Met Asp Tyr Tyr Asn 65
70 75 80 Val Asn Pro Glu Asp Leu Ile Val Leu Tyr Asp Asp Leu Asp
Leu Glu 85 90 95 Gln Gly Gln Val Arg Leu Arg Gln Lys Gly Ser Ala
Gly Gly His Asn 100 105 110 Gly Met Lys Ser Ile Ile Lys Met Leu Gly
Thr Asp Gln Phe Lys Arg 115 120 125 Ile Arg Ile Gly Val Gly Arg Pro
Thr Asn Gly Met Thr Val Pro Asp 130 135 140 Tyr Val Leu Gln Arg Phe
Ser Asn Asp Glu Met Val Thr Met Glu Lys 145 150 155 160 Val Ile Glu
His Ala Ala Arg Ala Ile Glu Lys Phe Val Glu Thr Ser 165 170 175 Arg
Phe Asp His Val Met Asn Glu Phe Asn Gly Glu Val Lys 180 185 190 33
1277 DNA Staphylococcus aureus 33 tgatccgatt atcttagtag gtgccaatga
aagttatgag ccacgttgtc gcgcgcacca 60 tatcgtagca cctagtgata
ataataagga ggaattataa gtgtttgatc aattagatat 120 tgtagaagaa
agatacgaac agttaaatga actgttaagt gacccagatg ttgtaaatga 180
ttcagataaa ttacgtaaat attctaaaga gcaagctgat ttacaaaaaa ctgtagatgt
240 ttatcgtaac tataaagcta aaaaagaaga attagctgat attgaagaaa
tgttaagtga 300 gactgatgat aaagaagaag tagaaatgtt aaaagaggag
agtaatggta ttaaagctga 360 acttccaaat cttgaagaag agcttaaaat
attattgatt cctaaagatc ctaatgatga 420 caaagacgtt attgtagaaa
taagagcagc agcaggtggt gatgaggctg cgatttttgc 480 tggtgattta
atgcgtatgt attcaaagta tgctgaatca caaggattca aaactgaaat 540
agtagaagcg tctgaaagtg accatggtgg ttacaaagaa attagtttct cagtttctgg
600 taatggcgcg tatagtaaat tgaaatttga aaatggtgcg caccgcgttc
aacgtgtgcc 660 tgaaacagaa tcaggtggac gtattcatac ttcaacagct
acagtggcag ttttaccaga 720 agttgaagat gtagaaattg aaattagaaa
tgaagattta aaaatcgaca cgtatcgttc 780 aagtggtgca ggtggtcagc
acgtaaacac aactgactct gcagtacgta ttacccattt 840 accaactggt
gtcattgcaa catcttctga gaagtctcaa attcaaaacc gtgaaaaagc 900
aatgaaagtg ttaaaagcac gtttatacga tatgaaagtt caagaagaac aacaaaagta
960 tgcgtcacaa cgtaaatcag cagtcggtac tggtgatcgt tcagaacgta
ttcgaactta 1020 taattatcca caaagccgtg taacagacca tcgtataggt
ctaacgcttc aaaaattagg 1080 gcaaattatg gaaggccatt tagaagaaat
tatagatgca ctgactttat cagagcagac 1140 agataaattg aaagaactta
ataatggtga attataaaga aaagttagat gaagcaattc 1200 atttaacaca
acaaaaaggg tttgaacaaa cacgagctga atggttaatg ttagatgtat 1260
ttcaatggac gcgtacg 1277 34 358 PRT Staphylococcus aureus 34 Val Phe
Asp Gln Leu Asp Ile Val Glu Glu Arg Tyr Glu Gln Leu Asn 1 5 10 15
Glu Leu Leu Ser Asp Pro Asp Val Val Asn Asp Ser Asp Lys Leu Arg 20
25 30 Lys Tyr Ser Lys Glu Gln Ala Asp Leu Gln Lys Thr Val Asp Val
Tyr 35 40 45 Arg Asn Tyr Lys Ala Lys Lys Glu Glu Leu Ala Asp Ile
Glu Glu Met 50 55 60 Leu Ser Glu Thr Asp Asp Lys Glu Glu Val Glu
Met Leu Lys Glu Glu 65 70 75 80 Ser Asn Gly Ile Lys Ala Glu Leu Pro
Asn Leu Glu Glu Glu Leu Lys 85 90 95 Ile Leu Leu Ile Pro Lys Asp
Pro Asn Asp Asp Lys Asp Val Ile Val 100 105 110 Glu Ile Arg Ala Ala
Ala Gly Gly Asp Glu Ala Ala Ile Phe Ala Gly 115 120 125 Asp Leu
Met
Arg Met Tyr Ser Lys Tyr Ala Glu Ser Gln Gly Phe Lys 130 135 140 Thr
Glu Ile Val Glu Ala Ser Glu Ser Asp His Gly Gly Tyr Lys Glu 145 150
155 160 Ile Ser Phe Ser Val Ser Gly Asn Gly Ala Tyr Ser Lys Leu Lys
Phe 165 170 175 Glu Asn Gly Ala His Arg Val Gln Arg Val Pro Glu Thr
Glu Ser Gly 180 185 190 Gly Arg Ile His Thr Ser Thr Ala Thr Val Ala
Val Leu Pro Glu Val 195 200 205 Glu Asp Val Glu Ile Glu Ile Arg Asn
Glu Asp Leu Lys Ile Asp Thr 210 215 220 Tyr Arg Ser Ser Gly Ala Gly
Gly Gln His Val Asn Thr Thr Asp Ser 225 230 235 240 Ala Val Arg Ile
Thr His Leu Pro Thr Gly Val Ile Ala Thr Ser Ser 245 250 255 Glu Lys
Ser Gln Ile Gln Asn Arg Glu Lys Ala Met Lys Val Leu Lys 260 265 270
Ala Arg Leu Tyr Asp Met Lys Val Gln Glu Glu Gln Gln Lys Tyr Ala 275
280 285 Ser Gln Arg Lys Ser Ala Val Gly Thr Gly Asp Arg Ser Glu Arg
Ile 290 295 300 Arg Thr Tyr Asn Tyr Pro Gln Ser Arg Val Thr Asp His
Arg Ile Gly 305 310 315 320 Leu Thr Leu Gln Lys Leu Gly Gln Ile Met
Glu Gly His Leu Glu Glu 325 330 335 Ile Ile Asp Ala Leu Thr Leu Ser
Glu Gln Thr Asp Lys Leu Lys Glu 340 345 350 Leu Asn Asn Gly Glu Leu
355 35 1315 DNA Staphylococcus aureus 35 atttcttaac attgttattt
aacaaaatta tgttaaaatt tagcattata aaagatgcaa 60 atcaatgact
tgaattgaaa tataaatagg agcgaatgct atggaattat cagaaatcaa 120
acgaaatata gataagtata atcaagattt aacacaaatt agggggtctc ttgacttaga
180 gaacaaagaa actaatattc aagaatatga agaaatgatg gcagaaccta
atttttggga 240 taaccaaacg aaagcgcaag atattataga taaaaataat
gcgttaaaag caatagttaa 300 tggttataaa acactacaag cagaagtaga
tgacatggat gctacttggg atttattaca 360 agaagaattt gatgaagaaa
tgaaagaaga cttagagcaa gaggtcatta attttaaggc 420 taaagtggat
gaatacgaat tgcaattatt attagatggg cctcacgatg ccaataacgc 480
aattctagag ttacatcctg gtgcaggtgg cacggagtct caagattggg ctaatatgct
540 atttagaatg tatcaacgtt attgtgagaa gaaaggcttt aaagttgaaa
ctgttgatta 600 tctacctggg gatgaagcgg ggattaaaag tgtaacattg
ctcatcaaag ggcataatgc 660 ttatggttat ttaaaagctg aaaaaggtgt
acaccgacta gtacgaattt ctccatttga 720 ttcatcagga cgtcgtcata
catcatttgc atcatgcgac gttattccag attttaataa 780 tgatgaaata
gagattgaaa tcaatccgga tgatattaca gttgatacat tcagagcttc 840
tggtgcaggt ggtcagcata ttaacaaaac tgaatcggca atacgaatta cccaccaccc
900 ctcaggtata gttgttaata accaaaatga acgttctcaa attaaaaacc
gtgaagcagc 960 tatgaaaatg ttaaagtcta aattatatca attaaaattg
gaagagcagg cacgtgaaat 1020 ggctgaaatt cgtggcgaac aaaaagaaat
cggctgggga agccaaatta gatcatatgt 1080 tttccatcca tactcaatgg
tgaaagatca tcgtacgaac gaagaaacag gtaaggttga 1140 tgcagtgatg
gatggagaca ttggaccatt tatcgaatca tatttaagac agacaatgtc 1200
gcacgattaa tatatatttt aaaaccgagg ctctaaaagg gcgtcggttt ttggtttttt
1260 taaaggtagc taaataaatt gtaaattaga ttttggaata tgatttgttt atgaa
1315 36 369 PRT Staphylococcus aureus 36 Met Glu Leu Ser Glu Ile
Lys Arg Asn Ile Asp Lys Tyr Asn Gln Asp 1 5 10 15 Leu Thr Gln Ile
Arg Gly Ser Leu Asp Leu Glu Asn Lys Glu Thr Asn 20 25 30 Ile Gln
Glu Tyr Glu Glu Met Met Ala Glu Pro Asn Phe Trp Asp Asn 35 40 45
Gln Thr Lys Ala Gln Asp Ile Ile Asp Lys Asn Asn Ala Leu Lys Ala 50
55 60 Ile Val Asn Gly Tyr Lys Thr Leu Gln Ala Glu Val Asp Asp Met
Asp 65 70 75 80 Ala Thr Trp Asp Leu Leu Gln Glu Glu Phe Asp Glu Glu
Met Lys Glu 85 90 95 Asp Leu Glu Gln Glu Val Ile Asn Phe Lys Ala
Lys Val Asp Glu Tyr 100 105 110 Glu Leu Gln Leu Leu Leu Asp Gly Pro
His Asp Ala Asn Asn Ala Ile 115 120 125 Leu Glu Leu His Pro Gly Ala
Gly Gly Thr Glu Ser Gln Asp Trp Ala 130 135 140 Asn Met Leu Phe Arg
Met Tyr Gln Arg Tyr Cys Glu Lys Lys Gly Phe 145 150 155 160 Lys Val
Glu Thr Val Asp Tyr Leu Pro Gly Asp Glu Ala Gly Ile Lys 165 170 175
Ser Val Thr Leu Leu Ile Lys Gly His Asn Ala Tyr Gly Tyr Leu Lys 180
185 190 Ala Glu Lys Gly Val His Arg Leu Val Arg Ile Ser Pro Phe Asp
Ser 195 200 205 Ser Gly Arg Arg His Thr Ser Phe Ala Ser Cys Asp Val
Ile Pro Asp 210 215 220 Phe Asn Asn Asp Glu Ile Glu Ile Glu Ile Asn
Pro Asp Asp Ile Thr 225 230 235 240 Val Asp Thr Phe Arg Ala Ser Gly
Ala Gly Gly Gln His Ile Asn Lys 245 250 255 Thr Glu Ser Ala Ile Arg
Ile Thr His His Pro Ser Gly Ile Val Val 260 265 270 Asn Asn Gln Asn
Glu Arg Ser Gln Ile Lys Asn Arg Glu Ala Ala Met 275 280 285 Lys Met
Leu Lys Ser Lys Leu Tyr Gln Leu Lys Leu Glu Glu Gln Ala 290 295 300
Arg Glu Met Ala Glu Ile Arg Gly Glu Gln Lys Glu Ile Gly Trp Gly 305
310 315 320 Ser Gln Ile Arg Ser Tyr Val Phe His Pro Tyr Ser Met Val
Lys Asp 325 330 335 His Arg Thr Asn Glu Glu Thr Gly Lys Val Asp Ala
Val Met Asp Gly 340 345 350 Asp Ile Gly Pro Phe Ile Glu Ser Tyr Leu
Arg Gln Thr Met Ser His 355 360 365 Asp 37 840 DNA Staphylococcus
aureus 37 aataactgaa aatatgatag aattggtaaa tgaatatctg gaaactggaa
tgatagttga 60 aggaattaaa aataataaaa ttttagttga ggatgaataa
aatgtcagct tttataactt 120 ttgagggccc agaaggctct ggaaaaacaa
ctgtaattaa tgaagtttac catagattag 180 taaaagatta tgatgtcatt
atgactagag aaccaggtgg tgttcctact ggtgaagaaa 240 tacgtaaaat
tgtattagaa ggcaatgata tggacattag aactgaagca atgttatttg 300
ctgcatctag aagagaacat cttgtattaa aggtcatacc agctttaaaa gaaggtaagg
360 ttgtgttgtg tgatcgctat atcgatagtt cattagctta tcaaggttat
gctagaggga 420 ttggcgttga agaagtaaga gcattaaacg aatttgcaat
aaatggatta tatccagact 480 tgacgattta tttaaatgtt agtgctgaag
taggtcgcga acgtattatt aaaaattcaa 540 gagatcaaaa tagattagat
caagaagatt taaagtttca cgaaaaagta attgaaggtt 600 accaagaaat
cattcataat gaatcacaac ggttcaaaag cgttaatgca gatcaacctc 660
ttgaaaatgt tgttgaagac acgtatcaaa ctatcatcaa atatttagaa aagatatgat
720 ataattgtta gaagaggtgt tataaaatga aaatgattat agcgatcgta
caagatcaag 780 atagtcagga acttgcagat caacttgtta aaaataactt
tagagcaaca aaattggcaa 840 38 205 PRT Staphylococcus aureus 38 Met
Ser Ala Phe Ile Thr Phe Glu Gly Pro Glu Gly Ser Gly Lys Thr 1 5 10
15 Thr Val Ile Asn Glu Val Tyr His Arg Leu Val Lys Asp Tyr Asp Val
20 25 30 Ile Met Thr Arg Glu Pro Gly Gly Val Pro Thr Gly Glu Glu
Ile Arg 35 40 45 Lys Ile Val Leu Glu Gly Asn Asp Met Asp Ile Arg
Thr Glu Ala Met 50 55 60 Leu Phe Ala Ala Ser Arg Arg Glu His Leu
Val Leu Lys Val Ile Pro 65 70 75 80 Ala Leu Lys Glu Gly Lys Val Val
Leu Cys Asp Arg Tyr Ile Asp Ser 85 90 95 Ser Leu Ala Tyr Gln Gly
Tyr Ala Arg Gly Ile Gly Val Glu Glu Val 100 105 110 Arg Ala Leu Asn
Glu Phe Ala Ile Asn Gly Leu Tyr Pro Asp Leu Thr 115 120 125 Ile Tyr
Leu Asn Val Ser Ala Glu Val Gly Arg Glu Arg Ile Ile Lys 130 135 140
Asn Ser Arg Asp Gln Asn Arg Leu Asp Gln Glu Asp Leu Lys Phe His 145
150 155 160 Glu Lys Val Ile Glu Gly Tyr Gln Glu Ile Ile His Asn Glu
Ser Gln 165 170 175 Arg Phe Lys Ser Val Asn Ala Asp Gln Pro Leu Glu
Asn Val Val Glu 180 185 190 Asp Thr Tyr Gln Thr Ile Ile Lys Tyr Leu
Glu Lys Ile 195 200 205 39 923 DNA Staphylococcus aureus 39
aatgttgctt tattaaaatg taaatcattc taataaaacg acaactgtgt cttctttact
60 tgtatatgtt acatatattc acgatagaga ggataagaaa atggctcaaa
tttctaaata 120 taaacgtgta gttttgaaac taagtggtga agcgttagct
ggagaaaaag gatttggcat 180 aaatccagta attattaaaa gtgttgctga
gcaagtggct gaagttgcta aaatggactg 240 tgaaatcgca gtaatcgttg
gtggcggaaa catttggaga ggtaaaacag gtagtgactt 300 aggtatggac
cgtggaactg ctgattacat gggtatgctt gcaactgtaa tgaatgcctt 360
agcattacaa gatagtttag aacaattgga ttgtgataca cgagtattaa catctattga
420 aatgaagcaa gtggctgaac cttatattcg tcgtcgtgca attagacact
tagaaaagaa 480 acgcgtagtt atttttgctg caggtattgg aaacccatac
ttctctacag atactacagc 540 ggcattacgt gctgcagaag ttgaagcaga
tgttatttta atgggcaaaa ataatgtaga 600 tggtgtatat tctgcagatc
ctaaagtaaa caaagatgcg gtaaaatatg aacatttaac 660 gcatattcaa
atgcttcaag aaggtttaca agtaatggat tcaacagcat cctcattctg 720
tatggataat aacattccgt taactgtttt ctctattatg gaagaaggaa atattaaacg
780 tgctgttatg ggtgaaaaga taggtacgtt aattacaaaa taaatttaga
ggtgtaaaat 840 aatgagtgac attattaatg aaactaaatc aagaatgcaa
aaatcaatcg aaagcttatc 900 acgtgaatta gctaacatca gtg 923 40 240 PRT
Staphylococcus aureus 40 Met Ala Gln Ile Ser Lys Tyr Lys Arg Val
Val Leu Lys Leu Ser Gly 1 5 10 15 Glu Ala Leu Ala Gly Glu Lys Gly
Phe Gly Ile Asn Pro Val Ile Ile 20 25 30 Lys Ser Val Ala Glu Gln
Val Ala Glu Val Ala Lys Met Asp Cys Glu 35 40 45 Ile Ala Val Ile
Val Gly Gly Gly Asn Ile Trp Arg Gly Lys Thr Gly 50 55 60 Ser Asp
Leu Gly Met Asp Arg Gly Thr Ala Asp Tyr Met Gly Met Leu 65 70 75 80
Ala Thr Val Met Asn Ala Leu Ala Leu Gln Asp Ser Leu Glu Gln Leu 85
90 95 Asp Cys Asp Thr Arg Val Leu Thr Ser Ile Glu Met Lys Gln Val
Ala 100 105 110 Glu Pro Tyr Ile Arg Arg Arg Ala Ile Arg His Leu Glu
Lys Lys Arg 115 120 125 Val Val Ile Phe Ala Ala Gly Ile Gly Asn Pro
Tyr Phe Ser Thr Asp 130 135 140 Thr Thr Ala Ala Leu Arg Ala Ala Glu
Val Glu Ala Asp Val Ile Leu 145 150 155 160 Met Gly Lys Asn Asn Val
Asp Gly Val Tyr Ser Ala Asp Pro Lys Val 165 170 175 Asn Lys Asp Ala
Val Lys Tyr Glu His Leu Thr His Ile Gln Met Leu 180 185 190 Gln Glu
Gly Leu Gln Val Met Asp Ser Thr Ala Ser Ser Phe Cys Met 195 200 205
Asp Asn Asn Ile Pro Leu Thr Val Phe Ser Ile Met Glu Glu Gly Asn 210
215 220 Ile Lys Arg Ala Val Met Gly Glu Lys Ile Gly Thr Leu Ile Thr
Lys 225 230 235 240 41 1013 DNA Staphylococcus aureus 41 gatagcatcc
atgtatagtg atagtattta caacaattat tataatacta tttagttaag 60
tagagaaata gttaaacatt tgaaagtgtg gtttaatgga atgtcagcaa taggaacagt
120 ttttaaagaa catgtaaaga acttttattt aattcaaaga ctggctcagt
ttcaagttaa 180 aattatcaat catagtaact atttaggtgt ggcttgggaa
ttaattaacc ctgttatgca 240 aattatggtt tactggatgg tttttggatt
aggaataaga agtaatgcac caattcatgg 300 tgtacctttt gtttattggt
tattggttgg tatcagtatg tggttcttca tcaaccaagg 360 tattttagaa
ggtactaaag caattacaca aaagtttaat caagtatcga aaatgaactt 420
cccgttatcg ataataccga catatattgt gacaagtaga ttttatggac atttaggctt
480 acttttactt gtgataattg catgtatgtt tactggtatt tatccatcaa
tacatatcat 540 tcaattattg atatatgtac cgttttgttt tttcttaact
gcctcggtga cgttattaac 600 atcaacactc ggtgtgttag ttagagatac
acaaatgtta atgcaagcaa tattaagaat 660 attattttac ttttcaccaa
ttttgtggct accaaagaac catggtatca gtggtttaat 720 tcatgaaatg
atgaaatata atccagttta ctttattgct gaatcatacc gtgcagcaat 780
tttatatcac gaatggtatt tcatggatca ttggaaatta atgttataca atttcggtat
840 tgttgccatt ttctttgcaa ttggtgcgta cttacacatg aaatatagag
atcaatttgc 900 agacttcttg taatatattt atatgacgaa accccgctaa
ccattaataa atggaagtgg 960 ggttcatttt tgtttataat ttaagtaaat
aacatattaa gttggtgtat tat 1013 42 270 PRT Staphylococcus aureus 42
Met Ser Ala Ile Gly Thr Val Phe Lys Glu His Val Lys Asn Phe Tyr 1 5
10 15 Leu Ile Gln Arg Leu Ala Gln Phe Gln Val Lys Ile Ile Asn His
Ser 20 25 30 Asn Tyr Leu Gly Val Ala Trp Glu Leu Ile Asn Pro Val
Met Gln Ile 35 40 45 Met Val Tyr Trp Met Val Phe Gly Leu Gly Ile
Arg Ser Asn Ala Pro 50 55 60 Ile His Gly Val Pro Phe Val Tyr Trp
Leu Leu Val Gly Ile Ser Met 65 70 75 80 Trp Phe Phe Ile Asn Gln Gly
Ile Leu Glu Gly Thr Lys Ala Ile Thr 85 90 95 Gln Lys Phe Asn Gln
Val Ser Lys Met Asn Phe Pro Leu Ser Ile Ile 100 105 110 Pro Thr Tyr
Ile Val Thr Ser Arg Phe Tyr Gly His Leu Gly Leu Leu 115 120 125 Leu
Leu Val Ile Ile Ala Cys Met Phe Thr Gly Ile Tyr Pro Ser Ile 130 135
140 His Ile Ile Gln Leu Leu Ile Tyr Val Pro Phe Cys Phe Phe Leu Thr
145 150 155 160 Ala Ser Val Thr Leu Leu Thr Ser Thr Leu Gly Val Leu
Val Arg Asp 165 170 175 Thr Gln Met Leu Met Gln Ala Ile Leu Arg Ile
Leu Phe Tyr Phe Ser 180 185 190 Pro Ile Leu Trp Leu Pro Lys Asn His
Gly Ile Ser Gly Leu Ile His 195 200 205 Glu Met Met Lys Tyr Asn Pro
Val Tyr Phe Ile Ala Glu Ser Tyr Arg 210 215 220 Ala Ala Ile Leu Tyr
His Glu Trp Tyr Phe Met Asp His Trp Lys Leu 225 230 235 240 Met Leu
Tyr Asn Phe Gly Ile Val Ala Ile Phe Phe Ala Ile Gly Ala 245 250 255
Tyr Leu His Met Lys Tyr Arg Asp Gln Phe Ala Asp Phe Leu 260 265 270
43 995 DNA Staphylococcus aureus 43 taacaaaatc ttctatacac
tttacaacag gttttaaaat ttaacaactg ttgagtagta 60 tattataatc
tagataaatg tgaataagga aggtctacaa atgaacgttt cggtaaacat 120
taaaaatgta acaaaagaat atcgtattta tcgtacaaat aaagaacgta tgaaagatgc
180 gctcattccc aaacataaaa acaaaacatt tttcgcttta gatgacatta
gtttaaaagc 240 atatgaaggt gacgtcatag ggcttgttgg catcaatggt
tccggcaaat caacgttgag 300 caatatcatt ggcggttctt tgtcgcctac
tgttggcaaa gtggatcgta atggtgaagt 360 cagcgttatc gcaattagtg
ctggcttgag tggacaactt acagggattg aaaatatcga 420 atttaaaatg
ttatgtatgg gctttaagcg aaaagaaatt aaagcgatga cacctaagat 480
tattgaattt agtgaacttg gtgagtttat ttatcaacca gttaaaaagt attcaagtgg
540 tatgcgtgca aaacttggtt tttcaattaa tatcacagtt aatccagata
tcttagtcat 600 tgacgaagct ttatctgtag gtgaccaaac ttttgcacaa
aaatgtttag ataaaattta 660 cgagtttaaa gagcaaaaca aaaccatctt
tttcgttagt cataacttag gacaagtgag 720 acaattttgt actaagattg
cttggattga aggcggaaag ttaaaagatt acggtgaact 780 tgatgatgta
ttacctaaat atgaagcttt ccttaacgat tttaaaaaga aatccaaagc 840
cgaacaaaaa gaatttagaa acaaactcga tgagtcccgc ttcgttatta aataaaccga
900 aaaaaccgag aatctccatt taaggatttc ctcggtttta tttttgtcat
catgattatt 960 tcgccttttt tatttttctt tttgctttgg ctatt 995 44 264
PRT Staphylococcus aureus 44 Met Asn Val Ser Val Asn Ile Lys Asn
Val Thr Lys Glu Tyr Arg Ile 1 5 10 15 Tyr Arg Thr Asn Lys Glu Arg
Met Lys Asp Ala Leu Ile Pro Lys His 20 25 30 Lys Asn Lys Thr Phe
Phe Ala Leu Asp Asp Ile Ser Leu Lys Ala Tyr 35 40 45 Glu Gly Asp
Val Ile Gly Leu Val Gly Ile Asn Gly Ser Gly Lys Ser 50 55 60 Thr
Leu Ser Asn Ile Ile Gly Gly Ser Leu Ser Pro Thr Val Gly Lys 65 70
75 80 Val Asp Arg Asn Gly Glu Val Ser Val Ile Ala Ile Ser Ala Gly
Leu 85 90 95 Ser Gly Gln Leu Thr Gly Ile Glu Asn Ile Glu Phe Lys
Met Leu Cys 100 105 110 Met Gly Phe Lys Arg Lys Glu Ile Lys Ala Met
Thr Pro Lys Ile Ile 115 120 125 Glu Phe Ser Glu Leu Gly Glu Phe Ile
Tyr Gln Pro Val Lys Lys Tyr 130 135 140 Ser Ser Gly Met Arg Ala Lys
Leu Gly Phe Ser Ile Asn Ile Thr Val 145 150 155 160 Asn Pro Asp Ile
Leu Val Ile Asp Glu Ala Leu Ser Val Gly Asp Gln 165 170 175 Thr Phe
Ala Gln Lys Cys Leu Asp Lys Ile Tyr Glu Phe Lys Glu Gln 180 185 190
Asn Lys Thr Ile Phe Phe Val Ser His Asn Leu Gly Gln Val Arg Gln 195
200 205 Phe Cys Thr Lys Ile Ala Trp Ile Glu Gly Gly Lys Leu Lys Asp
Tyr 210 215 220 Gly Glu Leu Asp Asp Val Leu Pro Lys Tyr Glu Ala Phe
Leu Asn Asp 225 230 235 240 Phe Lys Lys Lys Ser Lys Ala Glu Gln Lys
Glu Phe Arg Asn
Lys Leu 245 250 255 Asp Glu Ser Arg Phe Val Ile Lys 260 45 738 DNA
Staphylococcus aureus 45 ataaggtgaa gacacataaa acaatatatc
ttagtaagca tgcaacactc ttttttgttt 60 attcataaca acaaaaaaga
attaaaggag gagtcttatt atggctcgat tcagaggttc 120 aaactggaaa
aaatctcgtc gtttaggtat ctctttaagc ggtactggta aagaattaga 180
aaaacgtcct tacgcaccag gacaacatgg tccaaaccaa cgtaaaaaat tatcagaata
240 tggtttacaa ttacgtgaaa aacaaaaatt acgttactta tatggaatga
ctgaaagaca 300 attccgtaac acatttgaca tcgctggtaa aaaattcggt
gtacacggtg aaaacttcat 360 gatcttatta gcaagtcgtt tagacgctgt
tgtttattca ttaggtttag ctcgtactcg 420 tcgtcaagca cgtcaattag
ttaaccacgg tcatatctta gtagatggta aacgtgttga 480 tattccatct
tattctgtta aacctggtca aacaatttca gttcgtgaaa aatctcaaaa 540
attaaacatc atcgttgaat cagttgaaat caacaatttc gtacctgagt acttaaactt
600 tgatgctgac agcttaactg gtactttcgt acgtttacca gaacgtagcg
aattacctgc 660 tgaaattaac gaacaattaa tccgttgagt actactcaag
ataatacggt caataccaac 720 acccacaatt gtgggtgt 738 46 195 PRT
Staphylococcus aureus 46 Met Ala Arg Phe Arg Gly Ser Asn Trp Lys
Lys Ser Arg Arg Leu Gly 1 5 10 15 Ile Ser Leu Ser Gly Thr Gly Lys
Glu Leu Glu Lys Arg Pro Tyr Ala 20 25 30 Pro Gly Gln His Gly Pro
Asn Gln Arg Lys Lys Leu Ser Glu Tyr Gly 35 40 45 Leu Gln Leu Arg
Glu Lys Gln Lys Leu Arg Tyr Leu Tyr Gly Met Thr 50 55 60 Glu Arg
Gln Phe Arg Asn Thr Phe Asp Ile Ala Gly Lys Lys Phe Gly 65 70 75 80
Val His Gly Glu Asn Phe Met Ile Leu Leu Ala Ser Arg Leu Asp Ala 85
90 95 Val Val Tyr Ser Leu Gly Leu Ala Arg Thr Arg Arg Gln Ala Arg
Gln 100 105 110 Leu Val Asn His Gly His Ile Leu Val Asp Gly Lys Arg
Val Asp Ile 115 120 125 Pro Ser Tyr Ser Val Lys Pro Gly Gln Thr Ile
Ser Val Arg Glu Lys 130 135 140 Ser Gln Lys Leu Asn Ile Ile Val Glu
Ser Val Glu Ile Asn Asn Phe 145 150 155 160 Val Pro Glu Tyr Leu Asn
Phe Asp Ala Asp Ser Leu Thr Gly Thr Phe 165 170 175 Val Arg Leu Pro
Glu Arg Ser Glu Leu Pro Ala Glu Ile Asn Glu Gln 180 185 190 Leu Ile
Arg 195 47 980 DNA Staphylococcus aureus 47 tgttgattgc acctgcttca
gtcattgcta taactatttt aatttttaat ttaaccggtg 60 atgcactaag
agatagattg ctgaaacaac ggggtgaata tgatgagtct cattgatata 120
caaaatttaa caataaagaa tactagtgag aaatctctta ttaaagggat tgatttgaaa
180 atttttagtc aacagattaa tgccttgatt ggagagagcg gcgctggaaa
aagtttgatt 240 gctaaagctt tacttgaata tttaccattt gatttaagct
gcacgtatga ttcgtaccaa 300 tttgatgggg aaaatgttag tagattgagt
caatattatg gtcatacaat tggctatatt 360 tctcaaaatt atgcagaaag
ttttaacgac catactaaat taggtaaaca gttaactgcg 420 atttatcgta
agcattataa aggtagtaaa gaagaggctt tgtccaaagt tgataaggct 480
ttgtcgtggg ttaatttaca aagcaaagat atattaaata aatatagttt ccaactttct
540 gggggccaac ttgaacgcgt atacatagca agcgttctca tgttggagcc
taaattaatc 600 attgcagacg aaccagttgc atcattggat gctttgaacg
gtaatcaagt gatggattta 660 ttacagcata ttgtattaga acatggtcaa
acattattta ttatcacaca taacttaagt 720 catgtattga aatattgtca
gtacatttat gttttaaaag aaggtcaaat cattgaacga 780 ggtaatatta
atcatttcaa gtatgagcat ttgcatccgt atactgaacg tctaattaaa 840
tatagaacac aattaaagag ggattactat gattgagtta aaacatgtga cttttggtta
900 taataaaaag cagatggtgc tacaagatat caatattact atacctgatg
gagaaaatgt 960 tggtatttta ggcgaaagtg 980 48 258 PRT Staphylococcus
aureus 48 Met Met Ser Leu Ile Asp Ile Gln Asn Leu Thr Ile Lys Asn
Thr Ser 1 5 10 15 Glu Lys Ser Leu Ile Lys Gly Ile Asp Leu Lys Ile
Phe Ser Gln Gln 20 25 30 Ile Asn Ala Leu Ile Gly Glu Ser Gly Ala
Gly Lys Ser Leu Ile Ala 35 40 45 Lys Ala Leu Leu Glu Tyr Leu Pro
Phe Asp Leu Ser Cys Thr Tyr Asp 50 55 60 Ser Tyr Gln Phe Asp Gly
Glu Asn Val Ser Arg Leu Ser Gln Tyr Tyr 65 70 75 80 Gly His Thr Ile
Gly Tyr Ile Ser Gln Asn Tyr Ala Glu Ser Phe Asn 85 90 95 Asp His
Thr Lys Leu Gly Lys Gln Leu Thr Ala Ile Tyr Arg Lys His 100 105 110
Tyr Lys Gly Ser Lys Glu Glu Ala Leu Ser Lys Val Asp Lys Ala Leu 115
120 125 Ser Trp Val Asn Leu Gln Ser Lys Asp Ile Leu Asn Lys Tyr Ser
Phe 130 135 140 Gln Leu Ser Gly Gly Gln Leu Glu Arg Val Tyr Ile Ala
Ser Val Leu 145 150 155 160 Met Leu Glu Pro Lys Leu Ile Ile Ala Asp
Glu Pro Val Ala Ser Leu 165 170 175 Asp Ala Leu Asn Gly Asn Gln Val
Met Asp Leu Leu Gln His Ile Val 180 185 190 Leu Glu His Gly Gln Thr
Leu Phe Ile Ile Thr His Asn Leu Ser His 195 200 205 Val Leu Lys Tyr
Cys Gln Tyr Ile Tyr Val Leu Lys Glu Gly Gln Ile 210 215 220 Ile Glu
Arg Gly Asn Ile Asn His Phe Lys Tyr Glu His Leu His Pro 225 230 235
240 Tyr Thr Glu Arg Leu Ile Lys Tyr Arg Thr Gln Leu Lys Arg Asp Tyr
245 250 255 Tyr Asp 49 760 DNA Staphylococcus aureus misc_feature
(712)..(712) n equal a, t, c, or g 49 gatgatattt taattacaga
aaatggttgt caagtcttta ctaaatgcac aaaagacctt 60 atagttttaa
cataagcgtg taaaatgagg aggaaactga atgatttcgg ttaatgattt 120
taaaacaggt ttaacaattt ctgttgataa cgctatttgg aaagttatag acttccaaca
180 tgtaaagcct ggtaaaggtt cagcattcgt tcgttcaaaa ttacgtaatt
taagaactgg 240 tgcaattcaa gagaaaacgt ttagagctgg tgaaaaagtt
gaaccagcaa tgattgaaaa 300 tcgtcgcatg caatatttat atgctgacgg
rgataatcat gtatttatgg ataatgaaag 360 ctttgaacaa acagaacttt
caagtgatta cttaaaagaa gaattgaatt acttaaaaga 420 aggtatggaa
gtacaaattc aaacatacga aggtgaaact atcggtgttg aattacctaa 480
aactgttgaa ttaacagtaa ctgaaacaga acctggtatt aaaggtgata ctgcaactgg
540 tgccactaaa tcggcaactg ttgaaactgg ttatacatta aatgtacctt
tatttgtaaa 600 cgaaggtgac gttttaatta tcaacactgg tgatggaagc
tacatttcaa gaggataatc 660 tctaatttgt taacaaatag cttgtattca
ctatactgat ttaacgtaag anattctaaa 720 taagtctcat aaagctattg
cctaaaatga ttataggtta 760 50 185 PRT Staphylococcus aureus 50 Met
Ile Ser Val Asn Asp Phe Lys Thr Gly Leu Thr Ile Ser Val Asp 1 5 10
15 Asn Ala Ile Trp Lys Val Ile Asp Phe Gln His Val Lys Pro Gly Lys
20 25 30 Gly Ser Ala Phe Val Arg Ser Lys Leu Arg Asn Leu Arg Thr
Gly Ala 35 40 45 Ile Gln Glu Lys Thr Phe Arg Ala Gly Glu Lys Val
Glu Pro Ala Met 50 55 60 Ile Glu Asn Arg Arg Met Gln Tyr Leu Tyr
Ala Asp Gly Asp Asn His 65 70 75 80 Val Phe Met Asp Asn Glu Ser Phe
Glu Gln Thr Glu Leu Ser Ser Asp 85 90 95 Tyr Leu Lys Glu Glu Leu
Asn Tyr Leu Lys Glu Gly Met Glu Val Gln 100 105 110 Ile Gln Thr Tyr
Glu Gly Glu Thr Ile Gly Val Glu Leu Pro Lys Thr 115 120 125 Val Glu
Leu Thr Val Thr Glu Thr Glu Pro Gly Ile Lys Gly Asp Thr 130 135 140
Ala Thr Gly Ala Thr Lys Ser Ala Thr Val Glu Thr Gly Tyr Thr Leu 145
150 155 160 Asn Val Pro Leu Phe Val Asn Glu Gly Asp Val Leu Ile Ile
Asn Thr 165 170 175 Gly Asp Gly Ser Tyr Ile Ser Arg Gly 180 185 51
9326 DNA Staphylococcus aureus 51 ttaggatgta agaaagttcc agtgcaagaa
atccatgaaa cacaatattc aattagtaca 60 tggcaacata aagttccatt
tggtgtgtgg tgggaaacgt tacaacaaga acatcgcttg 120 ccatggacta
ctgagacaag acaagaagcg ccatttatta caatgtgtca tggtgataca 180
gaacaatatt tgtatacaaa agatttaggc gaagcacatt ttcaagtatg ggaaaaggtt
240 gtcgcaagtt atagtggttg ttgttctgta gagagaattg cacaaggtac
atatccttgt 300 ctttctcaac aagatgtact catgaagtat cagccattga
gttataagga aattgaagcg 360 gttgttcata aaggggaaac tgtgccagca
ggtgtgacac gctttaatat ttcaggacga 420 tgtcttaatc ttcaagtacc
actggcatta cttaaacaag atgatgatgt tgaacaatgc 480 gcaattggaa
gcagttttta gcagataagt ttgccaatat gagatgctat actgaaaaag 540
tatacttggt ggagcaatag ttttactgtg atgttgaggg aaatatgatg atttagcgta
600 ttgatagcga aaatataata aaacaatata gtgtggagaa cttttgatat
tttataaata 660 ttgaagttct ccatttttgt attttgcata taaaaattaa
ataaaataag gtatattaag 720 gtaaagtata aattttaaat aaatggggaa
tgagtatgag ctcaattata ggaaaaatag 780 caatttggat aggcatcgta
gctcaaatat attttagtgt cgtttttgtt aggatgatat 840 ctattaatat
tgctggagga tctgattacg aaacaatttt tttattagga ttaatattgg 900
ctcttttcac tgttttacca accatcttta ctgcgattta tatggaaagt tactctgtaa
960 tcggaggtgc actttttatt gtttatgcta ttattgcact gtgtttatat
aatttccttt 1020 cgtcaatttt atggctgatt ggtggtattt tgctgatttg
gaataaatac tcaaaagatg 1080 aatcgacaga cgaaaatgaa aaagttgata
ttgaaagtac agagaatcaa tttgaatcta 1140 aagataaaat cactaaagaa
taaagagaat atttaaggta aagtataaat tttaaataaa 1200 tggggaatag
acatggaaaa aaatgtagaa aaatcattca taaagatagg tttatatttt 1260
caaatagctt atatagtact catggctata actttatgtg ggtttgtaat ttgctatgga
1320 ctaattttcg gccttttcta tttattatca ggtagcagag ctgattattt
aatagtaaca 1380 atagttatat cggcaataat ttctatattt gtaattatac
tttcaatcgt acctgtcatc 1440 gtattggcat ctgacttatt taaagaaagg
atttcaaaag gtgtcatatt aattgtattg 1500 gctattatcg ctttagtatt
atgcaacttt gtatctgcaa tactctggtt tgtttcagcc 1560 atatctattt
taggtagaaa aaaattagta gctgcagcag atactaccac tattcaaaaa 1620
agtaaaggga acgcaaatca agcatcacat aaagacacgt gtaaaaagga acttgatagt
1680 caagacatga tggaacatcc tgaggttaaa aatcccacga ctaaaaacct
tgaaggattt 1740 aacgaagaaa tacataaaga tgaagctaca actaaagttg
tcagtgataa cacggaaccg 1800 cctattgaat caaaagacca tgtctcgaaa
aaagattgat gacaaactaa tcgagagact 1860 taaaaaaata atattcaaca
taagaacttt taaaacgaca tttaaacgca ttgccaatca 1920 ctaatggtag
tgcgtttaac tataccttaa atatctgaat attttgttaa atggagctac 1980
ctttgttgta ctattcaaat gaagaggagt aaaatgtaat taaaggaaag aaatttgagg
2040 agtgatcttt atgacaaaca acaaagtagc attagtaact ggcggagcac
aagggattgg 2100 ttttaaaatt gcagaacgtt tagtggaaga tggtttcaaa
gtagcagttg ttgatttcaa 2160 tgaagaaggg gcaaaagcag ctgcacttaa
attatcaagt gatggtacaa aagctattgc 2220 tatcaaagca gatgtatcaa
accgtgatga tgtatttaac gcataagaca aactgccgcg 2280 caatttggcg
atttccatgt catggttaac aatgccggcc ttggaccaac aacaccaatc 2340
gatacaatta ctgaagaaca gtttaaaaca gtatatggcg tgaacgttgc aggtgtgcta
2400 tggggtattc aagccgcaca tgaacaattt aaaaaattca atcatggcgg
taaaattatc 2460 aatgcaacat ctcaagcagg cgttgagggt aacccaggct
tgtctttata ttgcagtaca 2520 aaattcgcag tgcgaggttt aacacaagta
gccgcacaag atttagcgtc tgaaggtatt 2580 actgtgaatg cattcgcacc
tggtatcgtt caaacaccaa tgatggaaag tatcgcagtg 2640 gcaacagccg
aagaagcagg taaacctgaa gcatggggtt gggaacaatt tacaagtcag 2700
attgctttgg gcagagtttc tcaaccagaa gatgtttcaa atgtagtgag cttcttagct
2760 ggtaaagact ctgattacat tactggacaa acaattattg tagatggtgg
tatgagattc 2820 cgttaataat catccactaa tgataaataa atccttattg
ttaagtttaa tcacttagca 2880 gtaaggattt tttagtgcac ttagaaggga
gtgtattggt agaaaattaa taagcgaagt 2940 tcttaagtga gttatgatgt
cacagtctaa tgcatcagtt gaaagcatta ttagtattaa 3000 cacacccaag
atattataaa acatcacaaa aacaccacta tctaatttat ctcaataaaa 3060
attcacaaag ttatctcatt ttatttttat aaataaaaaa tatcgataaa aagcttacaa
3120 tactttatgt ttttatgata tatttttaat gtataaatga ggtggaagat
ttggaaagag 3180 ttttgataac tggtggggct ggttttattg ggtcgcattt
agtagatgat ttacaacaag 3240 attatgatgt ttatgttcta gataactata
gaacaggtaa acgagaaaat attaaaagtt 3300 tggctgacga tcatgtgttt
gaattagata ttcgtgaata tgatgcagtt gaacaaatca 3360 tgaagacata
tcaatttgat tatgttattc atttagcagc attagttagt gttgctgagt 3420
cggttgagaa acctatctta tctcaagaaa taaacgtcgt agcaacatta agattgttag
3480 aaatcattaa aaaatataat aatcatataa aacgttttat ctttgcttcg
tcagcagctg 3540 tttatggtga tcttcctgat ttgcctaaaa gtgatcaatc
attaatctta ccattatcac 3600 catatgcaat agataaatat tacggcgaac
ggacgacatt aaattattgt tcgttatata 3660 acataccaac agcggttgtt
aaatttttta atgtatttgg gccaagacag gatcctaagt 3720 cacaatattc
aggtgtgatt tcaaagatgt tcgattcatt tgagcataac aagccattta 3780
cattttttgg tgacggactg caaactagag attttgtata tgtatatgat gttgttcaat
3840 ctgtacgctt aattatggaa cacaaagatg caattggaca cggttataac
attggtacag 3900 gcacttttac taatttatta gaggtttatc gtattattgg
tgaattatat ggaaaatcag 3960 tcgagcatga atttaaagaa gcacgaaaag
gagatattaa gcattcttat gcagatattt 4020 ctaacttaaa ggcattagga
tttgttccta aatatacagt agaaacaggt ttaaaggatt 4080 actttaattt
tgaggtagat aatattgaag aagttacagc taaagaagtg gaaatgtcgt 4140
gaaaatgaca ttgaagctgt ccataataat aagggttatg cctatcaaag aaaattagac
4200 aaactagaag aagtgagaaa aagctattac ccaattaaac gtgcgattga
cttaatttta 4260 agcattgttt tattattttt aactttaccg attatggtta
tattcgccat tgctatcgtc 4320 atagattcgc caggaaaccc tatttatagt
caggttagag ttgggaagat gggtaaatta 4380 attaaaatat acaaattacg
ttcgatgtgc aaaaacgcag agaaaaacgg tgcgcaatgg 4440 gctgataaag
atgatgatcg tataacaaat gtcgggaagt ttattcgtaa aacacgcatt 4500
gatgaattac cacaactaat taatgttgtt aaaggggaaa tgagttttat tggaccacgc
4560 ccggaacgtc cggaatttgt agaattattt agttcagaag tgataggttt
cgagcaaaga 4620 tgtcttgtta caccagggtt aacaggactt gcgcaaattc
aaggtggata tgacttaaca 4680 ccgcaacaaa aactgaaata tgacatgaaa
tatatacata aaggtagttt aatgatggaa 4740 ctatatatat caattagaac
attgatggtt gttattacag gggaaggctc aaggtagtct 4800 taatttactt
aataagttca aataaaagtt atattttaaa gattgtgacc aattgttaca 4860
gtataacgag gaatcccttg agacagtatc aaatggcatt aagaaatatg tgccatcatt
4920 gatttgcatg gctataaata ctattcatct gatgagatag ccatgttaag
aaattgaaag 4980 tatagcatta aaggggtttg taacagttga aaattatata
ttgtattact aaagcagaca 5040 atggtggtgc acaaacacat ctcattcaac
tcgccaacca tttttgcgta cacaatgatg 5100 tttatgtcat tgtaggcaat
catggaccaa tgattgaaca actagatgca agagttaatg 5160 taattattat
cgaacattta gtaggtccaa ttgactttaa acaagatatt ttagctgtca 5220
aagtgttagc acagttattc tcgaaaatta aacctgatgt tatccattta cattcttcca
5280 aagctggaac ggtcggacga attgcgaagt tcatttcgaa atcgaaagac
acacgtatag 5340 tttttactgc acatggatgg gcttttacag agggtgttaa
accagctaaa aaatttctat 5400 atttagttat cgaaaaatta atgtcactta
ttacagatag cattatttgt gtttcagatt 5460 tcgataaaca gttagcgtta
aaatatcgat ttaatcgatt gaaattaacc acaatacata 5520 atggtattgc
agatgttccc gctgttaagc aaacgctaaa aagccaatca cataacaata 5580
ttggcgaagt agttggaatg ttgcctaata aacaagattt acagattaat gccccgacaa
5640 agcatcaatt tgttatgatt gcaagatttg cttatccaaa attgccacaa
aatctaatcg 5700 cggcaataga gatattgaaa ttacataaca gtaatcatgc
gcattttaca tttataggcg 5760 atggacctac attaaatgat tgtcagcaac
aagttgtaca agctgggtta gaaaatgatg 5820 tcacattttt gggcaatgtc
attaatgcga gtcatttatt atcacaatac gatacgttta 5880 ttttaataag
taagcatgaa ggtttgccaa ttagcattat agaagctatg gctacaggtt 5940
tgcctgttat agccagtcat gttggcggta tttcagaatt agtagctgat aatggtatat
6000 gtatgatgaa caaccaaccc gaaactattg ctaaagtcct ggaaaaatat
ttaatagaca 6060 gtgattacat caaaatgagt aatcaatcta gaaaacgtta
tttagaatgt tttactgagg 6120 agaaaatgat taaagaagtg gaagacgttt
ataatggaaa atcaacacaa tagtaaatta 6180 ctaacattgt tacttatcgg
tttagcggtt tttattcagc aatcttcggt tattgccggt 6240 gtgaatgttt
ctatagctga ctttatcaca ttactaatat tagtttattt actgtttttc 6300
gctaaccatt tattaaaggc aaatcatttt ttacagtttt tcattatttt gtatacatat
6360 cgtatgatta ttacgctttg tttgctattt tttgatgatt tgatatttat
tacggttaag 6420 gaagttcttg catctacagt taaatatgca tttgtagtca
tttatttcta tttagggatg 6480 atcatcttta agttaggtaa tagcaaaaaa
gtgatcgtta cctcttatat tataagcagt 6540 gtgactatag gtctattttg
tattatagct ggtttgaaca agtccccttt actaatgaaa 6600 ttgttatatt
ttgatgaaat acgttcaaaa ggattaatga atgaccctaa ctatttcgcg 6660
atgacacaga ttattacatt ggtacttgct tacaagtata ttcataatta catattcaag
6720 gtccttgcat gtggtatttt gctatggtct ttaactacaa cggggtctaa
gactgcgttt 6780 atcatattaa tcgtcttagc catttatttc tttattaaaa
agttatttag tagaaatgcg 6840 gtaagtgttg tgagtatgtc agtgattatg
ctgatattac tttgttttac cttttataat 6900 atcaactact atttattcca
attaagcgac cttgatgcct taccgtcatt agatcgaatg 6960 gcgtctattt
ttgaagaggg ctttgcatca ttaaatgata gtgggtctga gcgaagtgtt 7020
gtatggataa atgccatttc agtaattaaa tatacactag gttttggtgt cggattagtg
7080 gattatgtac atattggctc gcaaattaat ggtattttac ttgttgccca
taatacatat 7140 ttgcagatct ttgcggaatg gggcatttta ttcggtgcat
tatttatcat atttatgctt 7200 tatttactgt ttgaattatt tagatttaac
atttctggga aaaatgtaac agcaattgtt 7260 gtaatgttga cgatgctgat
ttacttttta acagtatcat ttaataactc aagatatgtc 7320 gcttttattt
taggaattat cgtctttatt gttcaatatg aaaagatgga aagggatcgt 7380
aatgaagagt gattcactaa aagaaaatat tatttatcaa gggctatacc aattgattag
7440 aacgatgaca ccactgatta caatacccat tatttcacgt gcatttggtc
ccagtggtgt 7500 gggtattgtt tcattttctt tcaatatcgt gcaatacttt
ttgatgattg caagtgttgg 7560 cgttcagtta tattttaata gagttatcgc
gaagtccgtt aacgacaaac ggcaattgtc 7620 acagcagttt tgggatatct
ttgtcagtaa attattttta gcgttaacag tttttgcgat 7680 gtatatggtc
gtaattacta tatttattga tgattactat cttattttcc tactacaagg 7740
aatctatatt ataggtgcag cactcgatat ttcatggttt tatgctggaa ctgaaaagtt
7800 taaaattcct agcctcagta atattgttgc gtctggtatt gtattaagtg
tagttgttat 7860 ttttgtcaaa gatcaatcag atttatcatt gtatgtattt
actattgcta ttgtgacggt 7920 attaaaccaa ttacctttgt ttatctattt
aaaacgatac attagctttg tttcggttaa 7980 ttggatacac gtctggcaat
tgtttcgttc gtcattagca tacttattac caaatggaca 8040 gctcaactta
tatactagta tttcttgcgt tgttcttggt ttagtaggta cataccaaca 8100
agttggtatc ttttctaacg catttaatat tttaacggtc gcaatcataa tgattaatac
8160 atttgatctt gtaatgattc cgcgtattac caaaatgtct
atccagcaat cacatagttt 8220 aactaaaacg ttagctaata atatgaatat
tcaattgata ttaacaatac ctatggtctt 8280 tggtttaatt gcaattatgc
catcatttta tttatggttc tttggtgagg aattcgcatc 8340 aactgtccca
ttgatgacca ttttagcgat acttgtatta atcattcctt taaatatgtt 8400
gataagcagg caatatttat taatagtgaa taaaataaga ttatataatg cgtcaattac
8460 tattggtgca gtgataaacc tagtattatg tattattttg atatattttt
atggaattta 8520 cggtgctgct attgcgcgtt taattacaga gtttttcttg
ctcatttggc gatttattga 8580 tattactaaa atcaatgtga agttgaatat
tgtaagtacg attcaatgtg tcattgctgc 8640 tgttatgatg tttattgtgc
ttggtgtggt caatcattat ttgcccccta caatgtacgc 8700 tacgctgcta
ttaattgcga ttggtatagt agtttatctt ttattaatga tgactatgaa 8760
aaatcaatac gtatggcaaa tattgaggca tcttcgacat aaaacaattt aagtaccggt
8820 aatgctatac tttagaaaat taagattaag aagaaaaggc aatttcttat
tgaaaaatgg 8880 aagttgtctt ttttaattct ctttaaaagc gggaaacaaa
agcagttaaa tgcctttttg 8940 cattcaatat taaatattat atcaatttcg
aatatttaaa ttttatataa ttggatataa 9000 caaataaata ataattattg
caaaacacac ccaaaattaa ttattataaa agtatattca 9060 taaaaggagg
aatatactta tggcatttaa attaccaaat ttaccatatg catatgatgc 9120
attggaacca tatatagatc aaagaacaat ggagtttcat cacgacaaac atcacaatac
9180 gtacgtgacg aaattaaacg caacagttga aggaacagag ttagagcatc
aatcactagc 9240 ggatatgatt gctaacttag acaaggtacc ggaagcgatg
gggtaccgag ctcgaattcg 9300 taatcatgtc atagctgttt cctgtg 9326 52 981
DNA Staphylococcus aureus 52 gtggaagatt tggaaagagt tttgataact
ggtggggctg gttttattgg gtcgcattta 60 gtagatgatt tacaacaaga
ttatgatgtt tatgttctag ataactatag aacaggtaaa 120 cgagaaaata
ttaaaagttt ggctgacgat catgtgtttg aattagatat tcgtgaatat 180
gatgcagttg aacaaatcat gaagacatat caatttgatt atgttattca tttagcagca
240 ttagttagtg ttgctgagtc ggttgagaaa cctatcttat ctcaagaaat
aaacgtcgta 300 gcaacattaa gattgttaga aatcattaaa aaatataata
atcatataaa acgttttatc 360 tttgcttcgt cagcagctgt ttatggtgat
cttcctgatt tgcctaaaag tgatcaatca 420 ttaatcttac cattatcacc
atatgcaata gataaatatt acggcgaacg gacgacatta 480 aattattgtt
cgttatataa cataccaaca gcggttgtta aattttttaa tgtatttggg 540
ccaagacagg atcctaagtc acaatattca ggtgtgattt caaagatgtt cgattcattt
600 gagcataaca agccatttac attttttggt gacggactgc aaactagaga
ttttgtatat 660 gtatatgatg ttgttcaatc tgtacgctta attatggaac
acaaagatgc aattggacac 720 ggttataaca ttggtacagg cacttttact
aatttattag aggtttatcg tattattggt 780 gaattatatg gaaaatcagt
cgagcatgaa tttaaagaag cacgaaaagg agatattaag 840 cattcttatg
cagatatttc taacttaaag gcattaggat ttgttcctaa atatacagta 900
gaaacaggtt taaaggatta ctttaatttt gaggtagata atattgaaga agttacagct
960 aaagaagtgg aaatgtcgtg a 981 53 326 PRT Staphylococcus aureus 53
Val Glu Asp Leu Glu Arg Val Leu Ile Thr Gly Gly Ala Gly Phe Ile 1 5
10 15 Gly Ser His Leu Val Asp Asp Leu Gln Gln Asp Tyr Asp Val Tyr
Val 20 25 30 Leu Asp Asn Tyr Arg Thr Gly Lys Arg Glu Asn Ile Lys
Ser Leu Ala 35 40 45 Asp Asp His Val Phe Glu Leu Asp Ile Arg Glu
Tyr Asp Ala Val Glu 50 55 60 Gln Ile Met Lys Thr Tyr Gln Phe Asp
Tyr Val Ile His Leu Ala Ala 65 70 75 80 Leu Val Ser Val Ala Glu Ser
Val Glu Lys Pro Ile Leu Ser Gln Glu 85 90 95 Ile Asn Val Val Ala
Thr Leu Arg Leu Leu Glu Ile Ile Lys Lys Tyr 100 105 110 Asn Asn His
Ile Lys Arg Phe Ile Phe Ala Ser Ser Ala Ala Val Tyr 115 120 125 Gly
Asp Leu Pro Asp Leu Pro Lys Ser Asp Gln Ser Leu Ile Leu Pro 130 135
140 Leu Ser Pro Tyr Ala Ile Asp Lys Tyr Tyr Gly Glu Arg Thr Thr Leu
145 150 155 160 Asn Tyr Cys Ser Leu Tyr Asn Ile Pro Thr Ala Val Val
Lys Phe Phe 165 170 175 Asn Val Phe Gly Pro Arg Gln Asp Pro Lys Ser
Gln Tyr Ser Gly Val 180 185 190 Ile Ser Lys Met Phe Asp Ser Phe Glu
His Asn Lys Pro Phe Thr Phe 195 200 205 Phe Gly Asp Gly Leu Gln Thr
Arg Asp Phe Val Tyr Val Tyr Asp Val 210 215 220 Val Gln Ser Val Arg
Leu Ile Met Glu His Lys Asp Ala Ile Gly His 225 230 235 240 Gly Tyr
Asn Ile Gly Thr Gly Thr Phe Thr Asn Leu Leu Glu Val Tyr 245 250 255
Arg Ile Ile Gly Glu Leu Tyr Gly Lys Ser Val Glu His Glu Phe Lys 260
265 270 Glu Ala Arg Lys Gly Asp Ile Lys His Ser Tyr Ala Asp Ile Ser
Asn 275 280 285 Leu Lys Ala Leu Gly Phe Val Pro Lys Tyr Thr Val Glu
Thr Gly Leu 290 295 300 Lys Asp Tyr Phe Asn Phe Glu Val Asp Asn Ile
Glu Glu Val Thr Ala 305 310 315 320 Lys Glu Val Glu Met Ser 325 54
504 DNA Staphylococcus aureus 54 atggttatat tcgccattgc tatcgtcata
gattcgccag gaaaccctat ttatagtcag 60 gttagagttg ggaagatggg
taaattaatt aaaatataca aattacgttc gatgtgcaaa 120 aacgcagaga
aaaacggtgc gcaatgggct gataaagatg atgatcgtat aacaaatgtc 180
gggaagttta ttcgtaaaac acgcattgat gaattaccac aactaattaa tgttgttaaa
240 ggggaaatga gttttattgg accacgcccg gaacgtccgg aatttgtaga
attatttagt 300 tcagaagtga taggtttcga gcaaagatgt cttgttacac
cagggttaac aggacttgcg 360 caaattcaag gtggatatga cttaacaccg
caacaaaaac tgaaatatga catgaaatat 420 atacataaag gtagtttaat
gatggaacta tatatatcaa ttagaacatt gatggttgtt 480 attacagggg
aaggctcaag gtag 504 55 200 PRT Staphylococcus aureus 55 Leu Asp Lys
Leu Glu Glu Val Arg Lys Ser Tyr Tyr Pro Ile Lys Arg 1 5 10 15 Ala
Ile Asp Leu Ile Leu Ser Ile Val Leu Leu Phe Leu Thr Leu Pro 20 25
30 Ile Met Val Ile Phe Ala Ile Ala Ile Val Ile Asp Ser Pro Gly Asn
35 40 45 Pro Ile Tyr Ser Gln Val Arg Val Gly Lys Met Gly Lys Leu
Ile Lys 50 55 60 Ile Tyr Lys Leu Arg Ser Met Cys Lys Asn Ala Glu
Lys Asn Gly Ala 65 70 75 80 Gln Trp Ala Asp Lys Asp Asp Asp Arg Ile
Thr Asn Val Gly Lys Phe 85 90 95 Ile Arg Lys Thr Arg Ile Asp Glu
Leu Pro Gln Leu Ile Asn Val Val 100 105 110 Lys Gly Glu Met Ser Phe
Ile Gly Pro Arg Pro Glu Arg Pro Glu Phe 115 120 125 Val Glu Leu Phe
Ser Ser Glu Val Ile Gly Phe Glu Gln Arg Cys Leu 130 135 140 Val Thr
Pro Gly Leu Thr Gly Leu Ala Gln Ile Gln Gly Gly Tyr Asp 145 150 155
160 Leu Thr Pro Gln Gln Lys Leu Lys Tyr Asp Met Lys Tyr Ile His Lys
165 170 175 Gly Ser Leu Met Met Glu Leu Tyr Ile Ser Ile Arg Thr Leu
Met Val 180 185 190 Val Ile Thr Gly Glu Gly Ser Arg 195 200 56 1044
DNA Staphylococcus aureus 56 atgattgaac aactagatgc aagagttaat
gtaattatta tcgaacattt agtaggtcca 60 attgacttta aacaagatat
tttagctgtc aaagtgttag cacagttatt ctcgaaaatt 120 aaacctgatg
ttatccattt acattcttcc aaagctggaa cggtcggacg aattgcgaag 180
ttcatttcga aatcgaaaga cacacgtata gtttttactg cacatggatg ggcttttaca
240 gagggtgtta aaccagctaa aaaatttcta tatttagtta tcgaaaaatt
aatgtcactt 300 attacagata gcattatttg tgtttcagat ttcgataaac
agttagcgtt aaaatatcga 360 tttaatcgat tgaaattaac cacaatacat
aatggtattg cagatgttcc cgctgttaag 420 caaacgctaa aaagccaatc
acataacaat attggcgaag tagttggaat gttgcctaat 480 aaacaagatt
tacagattaa tgccccgaca aagcatcaat ttgttatgat tgcaagattt 540
gcttatccaa aattgccaca aaatctaatc gcggcaatag agatattgaa attacataac
600 agtaatcatg cgcattttac atttataggc gatggaccta cattaaatga
ttgtcagcaa 660 caagttgtac aagctgggtt agaaaatgat gtcacatttt
tgggcaatgt cattaatgcg 720 agtcatttat tatcacaata cgatacgttt
attttaataa gtaagcatga aggtttgcca 780 attagcatta tagaagctat
ggctacaggt ttgcctgtta tagccagtca tgttggcggt 840 atttcagaat
tagtagctga taatggtata tgtatgatga acaaccaacc cgaaactatt 900
gctaaagtcc tggaaaaata tttaatagac agtgattaca tcaaaatgag taatcaatct
960 agaaaacgtt atttagaatg ttttactgag gagaaaatga ttaaagaagt
ggaagacgtt 1020 tataatggaa aatcaacaca atag 1044 57 388 PRT
Staphylococcus aureus 57 Leu Lys Ile Ile Tyr Cys Ile Thr Lys Ala
Asp Asn Gly Gly Ala Gln 1 5 10 15 Thr His Leu Ile Gln Leu Ala Asn
His Phe Cys Val His Asn Asp Val 20 25 30 Tyr Val Ile Val Gly Asn
His Gly Pro Met Ile Glu Gln Leu Asp Ala 35 40 45 Arg Val Asn Val
Ile Ile Ile Glu His Leu Val Gly Pro Ile Asp Phe 50 55 60 Lys Gln
Asp Ile Leu Ala Val Lys Val Leu Ala Gln Leu Phe Ser Lys 65 70 75 80
Ile Lys Pro Asp Val Ile His Leu His Ser Ser Lys Ala Gly Thr Val 85
90 95 Gly Arg Ile Ala Lys Phe Ile Ser Lys Ser Lys Asp Thr Arg Ile
Val 100 105 110 Phe Thr Ala His Gly Trp Ala Phe Thr Glu Gly Val Lys
Pro Ala Lys 115 120 125 Lys Phe Leu Tyr Leu Val Ile Glu Lys Leu Met
Ser Leu Ile Thr Asp 130 135 140 Ser Ile Ile Cys Val Ser Asp Phe Asp
Lys Gln Leu Ala Leu Lys Tyr 145 150 155 160 Arg Phe Asn Arg Leu Lys
Leu Thr Thr Ile His Asn Gly Ile Ala Asp 165 170 175 Val Pro Ala Val
Lys Gln Thr Leu Lys Ser Gln Ser His Asn Asn Ile 180 185 190 Gly Glu
Val Val Gly Met Leu Pro Asn Lys Gln Asp Leu Gln Ile Asn 195 200 205
Ala Pro Thr Lys His Gln Phe Val Met Ile Ala Arg Phe Ala Tyr Pro 210
215 220 Lys Leu Pro Gln Asn Leu Ile Ala Ala Ile Glu Ile Leu Lys Leu
His 225 230 235 240 Asn Ser Asn His Ala His Phe Thr Phe Ile Gly Asp
Gly Pro Thr Leu 245 250 255 Asn Asp Cys Gln Gln Gln Val Val Gln Ala
Gly Leu Glu Asn Asp Val 260 265 270 Thr Phe Leu Gly Asn Val Ile Asn
Ala Ser His Leu Leu Ser Gln Tyr 275 280 285 Asp Thr Phe Ile Leu Ile
Ser Lys His Glu Gly Leu Pro Ile Ser Ile 290 295 300 Ile Glu Ala Met
Ala Thr Gly Leu Pro Val Ile Ala Ser His Val Gly 305 310 315 320 Gly
Ile Ser Glu Leu Val Ala Asp Asn Gly Ile Cys Met Met Asn Asn 325 330
335 Gln Pro Glu Thr Ile Ala Lys Val Leu Glu Lys Tyr Leu Ile Asp Ser
340 345 350 Asp Tyr Ile Lys Met Ser Asn Gln Ser Arg Lys Arg Tyr Leu
Glu Cys 355 360 365 Phe Thr Glu Glu Lys Met Ile Lys Glu Val Glu Asp
Val Tyr Asn Gly 370 375 380 Lys Ser Thr Gln 385 58 1239 DNA
Staphylococcus aureus 58 atggaaaatc aacacaatag taaattacta
acattgttac ttatcggttt agcggttttt 60 attcagcaat cttcggttat
tgccggtgtg aatgtttcta tagctgactt tatcacatta 120 ctaatattag
tttatttact gtttttcgct aaccatttat taaaggcaaa tcatttttta 180
cagtttttca ttattttgta tacatatcgt atgattatta cgctttgttt gctatttttt
240 gatgatttga tatttattac ggttaaggaa gttcttgcat ctacagttaa
atatgcattt 300 gtagtcattt atttctattt agggatgatc atctttaagt
taggtaatag caaaaaagtg 360 atcgttacct cttatattat aagcagtgtg
actataggtc tattttgtat tatagctggt 420 ttgaacaagt cccctttact
aatgaaattg ttatattttg atgaaatacg ttcaaaagga 480 ttaatgaatg
accctaacta tttcgcgatg acacagatta ttacattggt acttgcttac 540
aagtatattc ataattacat attcaaggtc cttgcatgtg gtattttgct atggtcttta
600 actacaacgg ggtctaagac tgcgtttatc atattaatcg tcttagccat
ttatttcttt 660 attaaaaagt tatttagtag aaatgcggta agtgttgtga
gtatgtcagt gattatgctg 720 atattacttt gttttacctt ttataatatc
aactactatt tattccaatt aagcgacctt 780 gatgccttac cgtcattaga
tcgaatggcg tctatttttg aagagggctt tgcatcatta 840 aatgatagtg
ggtctgagcg aagtgttgta tggataaatg ccatttcagt aattaaatat 900
acactaggtt ttggtgtcgg attagtggat tatgtacata ttggctcgca aattaatggt
960 attttacttg ttgcccataa tacatatttg cagatctttg cggaatgggg
cattttattc 1020 ggtgcattat ttatcatatt tatgctttat ttactgtttg
aattatttag atttaacatt 1080 tctgggaaaa atgtaacagc aattgttgta
atgttgacga tgctgattta ctttttaaca 1140 gtatcattta ataactcaag
atatgtcgct tttattttag gaattatcgt ctttattgtt 1200 caatatgaaa
agatggaaag ggatcgtaat gaagagtga 1239 59 412 PRT Staphylococcus
aureus 59 Met Glu Asn Gln His Asn Ser Lys Leu Leu Thr Leu Leu Leu
Ile Gly 1 5 10 15 Leu Ala Val Phe Ile Gln Gln Ser Ser Val Ile Ala
Gly Val Asn Val 20 25 30 Ser Ile Ala Asp Phe Ile Thr Leu Leu Ile
Leu Val Tyr Leu Leu Phe 35 40 45 Phe Ala Asn His Leu Leu Lys Ala
Asn His Phe Leu Gln Phe Phe Ile 50 55 60 Ile Leu Tyr Thr Tyr Arg
Met Ile Ile Thr Leu Cys Leu Leu Phe Phe 65 70 75 80 Asp Asp Leu Ile
Phe Ile Thr Val Lys Glu Val Leu Ala Ser Thr Val 85 90 95 Lys Tyr
Ala Phe Val Val Ile Tyr Phe Tyr Leu Gly Met Ile Ile Phe 100 105 110
Lys Leu Gly Asn Ser Lys Lys Val Ile Val Thr Ser Tyr Ile Ile Ser 115
120 125 Ser Val Thr Ile Gly Leu Phe Cys Ile Ile Ala Gly Leu Asn Lys
Ser 130 135 140 Pro Leu Leu Met Lys Leu Leu Tyr Phe Asp Glu Ile Arg
Ser Lys Gly 145 150 155 160 Leu Met Asn Asp Pro Asn Tyr Phe Ala Met
Thr Gln Ile Ile Thr Leu 165 170 175 Val Leu Ala Tyr Lys Tyr Ile His
Asn Tyr Ile Phe Lys Val Leu Ala 180 185 190 Cys Gly Ile Leu Leu Trp
Ser Leu Thr Thr Thr Gly Ser Lys Thr Ala 195 200 205 Phe Ile Ile Leu
Ile Val Leu Ala Ile Tyr Phe Phe Ile Lys Lys Leu 210 215 220 Phe Ser
Arg Asn Ala Val Ser Val Val Ser Met Ser Val Ile Met Leu 225 230 235
240 Ile Leu Leu Cys Phe Thr Phe Tyr Asn Ile Asn Tyr Tyr Leu Phe Gln
245 250 255 Leu Ser Asp Leu Asp Ala Leu Pro Ser Leu Asp Arg Met Ala
Ser Ile 260 265 270 Phe Glu Glu Gly Phe Ala Ser Leu Asn Asp Ser Gly
Ser Glu Arg Ser 275 280 285 Val Val Trp Ile Asn Ala Ile Ser Val Ile
Lys Tyr Thr Leu Gly Phe 290 295 300 Gly Val Gly Leu Val Asp Tyr Val
His Ile Gly Ser Gln Ile Asn Gly 305 310 315 320 Ile Leu Leu Val Ala
His Asn Thr Tyr Leu Gln Ile Phe Ala Glu Trp 325 330 335 Gly Ile Leu
Phe Gly Ala Leu Phe Ile Ile Phe Met Leu Tyr Leu Leu 340 345 350 Phe
Glu Leu Phe Arg Phe Asn Ile Ser Gly Lys Asn Val Thr Ala Ile 355 360
365 Val Val Met Leu Thr Met Leu Ile Tyr Phe Leu Thr Val Ser Phe Asn
370 375 380 Asn Ser Arg Tyr Val Ala Phe Ile Leu Gly Ile Ile Val Phe
Ile Val 385 390 395 400 Gln Tyr Glu Lys Met Glu Arg Asp Arg Asn Glu
Glu 405 410 60 1455 DNA Staphylococcus aureus 60 atgaaaagat
ggaaagggat cgtaatgaag agtgattcac taaaagaaaa tattatttat 60
caagggctat accaattgat tagaacgatg acaccactga ttacaatacc cattatttca
120 cgtgcatttg gtcccagtgg tgtgggtatt gtttcatttt ctttcaatat
cgtgcaatac 180 tttttgatga ttgcaagtgt tggcgttcag ttatatttta
atagagttat cgcgaagtcc 240 gttaacgaca aacggcaatt gtcacagcag
ttttgggata tctttgtcag taaattattt 300 ttagcgttaa cagtttttgc
gatgtatatg gtcgtaatta ctatatttat tgatgattac 360 tatcttattt
tcctactaca aggaatctat attataggtg cagcactcga tatttcatgg 420
ttttatgctg gaactgaaaa gtttaaaatt cctagcctca gtaatattgt tgcgtctggt
480 attgtattaa gtgtagttgt tatttttgtc aaagatcaat cagatttatc
attgtatgta 540 tttactattg ctattgtgac ggtattaaac caattacctt
tgtttatcta tttaaaacga 600 tacattagct ttgtttcggt taattggata
cacgtctggc aattgtttcg ttcgtcatta 660 gcatacttat taccaaatgg
acagctcaac ttatatacta gtatttcttg cgttgttctt 720 ggtttagtag
gtacatacca acaagttggt atcttttcta acgcatttaa tattttaacg 780
gtcgcaatca taatgattaa tacatttgat cttgtaatga ttccgcgtat taccaaaatg
840 tctatccagc aatcacatag tttaactaaa acgttagcta ataatatgaa
tattcaattg 900 atattaacaa tacctatggt ctttggttta attgcaatta
tgccatcatt ttatttatgg 960 ttctttggtg aggaattcgc atcaactgtc
ccattgatga ccattttagc gatacttgta 1020 ttaatcattc ctttaaatat
gttgataagc aggcaatatt tattaatagt gaataaaata 1080 agattatata
atgcgtcaat tactattggt gcagtgataa acctagtatt atgtattatt 1140
ttgatatatt tttatggaat ttacggtgct gctattgcgc gtttaattac agagtttttc
1200 ttgctcattt ggcgatttat tgatattact aaaatcaatg tgaagttgaa
tattgtaagt 1260 acgattcaat gtgtcattgc tgctgttatg atgtttattg
tgcttggtgt ggtcaatcat 1320 tatttgcccc ctacaatgta cgctacgctg
ctattaattg cgattggtat agtagtttat 1380 cttttattaa tgatgactat
gaaaaatcaa tacgtatggc aaatattgag gcatcttcga 1440 cataaaacaa tttaa
1455 61 476 PRT Staphylococcus aureus 61 Met Lys Ser Asp Ser Leu
Lys Glu Asn Ile Ile Tyr
Gln Gly Leu Tyr 1 5 10 15 Gln Leu Ile Arg Thr Met Thr Pro Leu Ile
Thr Ile Pro Ile Ile Ser 20 25 30 Arg Ala Phe Gly Pro Ser Gly Val
Gly Ile Val Ser Phe Ser Phe Asn 35 40 45 Ile Val Gln Tyr Phe Leu
Met Ile Ala Ser Val Gly Val Gln Leu Tyr 50 55 60 Phe Asn Arg Val
Ile Ala Lys Ser Val Asn Asp Lys Arg Gln Leu Ser 65 70 75 80 Gln Gln
Phe Trp Asp Ile Phe Val Ser Lys Leu Phe Leu Ala Leu Thr 85 90 95
Val Phe Ala Met Tyr Met Val Val Ile Thr Ile Phe Ile Asp Asp Tyr 100
105 110 Tyr Leu Ile Phe Leu Leu Gln Gly Ile Tyr Ile Ile Gly Ala Ala
Leu 115 120 125 Asp Ile Ser Trp Phe Tyr Ala Gly Thr Glu Lys Phe Lys
Ile Pro Ser 130 135 140 Leu Ser Asn Ile Val Ala Ser Gly Ile Val Leu
Ser Val Val Val Ile 145 150 155 160 Phe Val Lys Asp Gln Ser Asp Leu
Ser Leu Tyr Val Phe Thr Ile Ala 165 170 175 Ile Val Thr Val Leu Asn
Gln Leu Pro Leu Phe Ile Tyr Leu Lys Arg 180 185 190 Tyr Ile Ser Phe
Val Ser Val Asn Trp Ile His Val Trp Gln Leu Phe 195 200 205 Arg Ser
Ser Leu Ala Tyr Leu Leu Pro Asn Gly Gln Leu Asn Leu Tyr 210 215 220
Thr Ser Ile Ser Cys Val Val Leu Gly Leu Val Gly Thr Tyr Gln Gln 225
230 235 240 Val Gly Ile Phe Ser Asn Ala Phe Asn Ile Leu Thr Val Ala
Ile Ile 245 250 255 Met Ile Asn Thr Phe Asp Leu Val Met Ile Pro Arg
Ile Thr Lys Met 260 265 270 Ser Ile Gln Gln Ser His Ser Leu Thr Lys
Thr Leu Ala Asn Asn Met 275 280 285 Asn Ile Gln Leu Ile Leu Thr Ile
Pro Met Val Phe Gly Leu Ile Ala 290 295 300 Ile Met Pro Ser Phe Tyr
Leu Trp Phe Phe Gly Glu Glu Phe Ala Ser 305 310 315 320 Thr Val Pro
Leu Met Thr Ile Leu Ala Ile Leu Val Leu Ile Ile Pro 325 330 335 Leu
Asn Met Leu Ile Ser Arg Gln Tyr Leu Leu Ile Val Asn Lys Ile 340 345
350 Arg Leu Tyr Asn Ala Ser Ile Thr Ile Gly Ala Val Ile Asn Leu Val
355 360 365 Leu Cys Ile Ile Leu Ile Tyr Phe Tyr Gly Ile Tyr Gly Ala
Ala Ile 370 375 380 Ala Arg Leu Ile Thr Glu Phe Phe Leu Leu Ile Trp
Arg Phe Ile Asp 385 390 395 400 Ile Thr Lys Ile Asn Val Lys Leu Asn
Ile Val Ser Thr Ile Gln Cys 405 410 415 Val Ile Ala Ala Val Met Met
Phe Ile Val Leu Gly Val Val Asn His 420 425 430 Tyr Leu Pro Pro Thr
Met Tyr Ala Thr Leu Leu Leu Ile Ala Ile Gly 435 440 445 Ile Val Val
Tyr Leu Leu Leu Met Met Thr Met Lys Asn Gln Tyr Val 450 455 460 Trp
Gln Ile Leu Arg His Leu Arg His Lys Thr Ile 465 470 475
* * * * *