U.S. patent application number 11/439061 was filed with the patent office on 2006-11-30 for anti-adhesin based passive immunoprophlactic.
Invention is credited to Stephen J. Savarino.
Application Number | 20060269560 11/439061 |
Document ID | / |
Family ID | 37452779 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269560 |
Kind Code |
A1 |
Savarino; Stephen J. |
November 30, 2006 |
Anti-adhesin based passive immunoprophlactic
Abstract
The invention relates to an immunogenic composition and method
of the immunogenic composition for the production and
administration of a passive immunoprophylactic against
enterotoxigenic Escherichia coli. The immunoprophylactic is made
collecting anti-adhesin in the colostrum or milk of vaccinated
domesticated animals such as cows. The immunoprophylactic is
administered either as a dietary supplement or in capsular or
tablet form.
Inventors: |
Savarino; Stephen J.;
(Kensington, MD) |
Correspondence
Address: |
NAVAL MEDICAL RESEARCH CENTER;ATTN: (CODE 00L)
503 ROBERT GRANT AVENUE
SILVER SPRING
MD
20910-7500
US
|
Family ID: |
37452779 |
Appl. No.: |
11/439061 |
Filed: |
May 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60683787 |
May 24, 2005 |
|
|
|
Current U.S.
Class: |
424/169.1 ;
424/535 |
Current CPC
Class: |
Y02A 50/30 20180101;
C07K 16/1232 20130101; A61P 31/04 20180101; A61P 1/12 20180101;
C07K 2317/76 20130101; Y02A 50/474 20180101; A61K 2039/505
20130101; A61K 39/0258 20130101 |
Class at
Publication: |
424/169.1 ;
424/535 |
International
Class: |
A61K 39/40 20060101
A61K039/40; A61K 35/20 20060101 A61K035/20 |
Claims
1. A pharmaceutical composition, wherein said composition is
composed of colostrums or milk immunoglobulin against
enterotoxigenic Escherichia coli adhesin molecules.
2. The pharmaceutical composition of claim 1, wherein said adhesin
molecules are derived from Class 5 fimbriae.
3. The pharmaceutical composition of claim 1, wherein the said
adhesin molecule is one or more fimbrial minor subunits selected
from the group consisting of CfaE, CsfD, CsuD, CooD, CosD, CsdD,
CsbD and CotD.
4. A method of producing the anti-enterotoxigenic Escherichia coli
composition of claim 1 comprising the steps: a. administering to a
milk producing domesticated animal an immunogen composed of one or
more Class five Escherichia coli fimbrial adhesins or fragments
thereof; b. collecting immunoglobulin containing colostrum or milk
from said domesticated animal.
5. The method of claim 4, wherein the concentration of anti-adhesin
immunoglobulin in said collected colostrum or milk is adjusted to
0.1 g IgG per dose to 20.0 g of IgG per dose of said passive
prophylactic.
6. The method of claim 4, wherein said domesticated animal is a cow
or goat.
7. The method of claim 4, wherein said adhesin is selected from the
group consisting of CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and
CotD.
8. The method of claim 4, wherein said immunogen also comprises one
or more Escherichia coli major fimbrial subunit is selected from
the group consisting of CfaB, CsfA, CsuA1, CsuA2, CooA, CosA, CsbA,
CsdA and CotB.
9. The method of claim 4, wherein said immunogen is an Escherichia
coli fimbrial adhesin domain and polyhistidine tail fusion
polypeptide composed of the amino acid sequence selected from the
group consisting of SEQ ID No. 35, SEQ ID No. 36 and SEQ ID No.
37.
10. The method of claim 8, wherein said Escherichia coli fimbrial
adhesin is linked at its C-terminus to a linker which is
operatively linked at its C-terminus to said Escherichia coli major
fimbrial subunit.
11. The method of claim 10, wherein said Escherichia coli fimbrial
adhesin is a monomer or polymer of adhesin polypeptides.
12. The method of claim 10, wherein said linker is composed of the
amino acid sequence selected from the group consisting of SEQ ID
No. 10, 12 and 13.
13. The method of claim 10, wherein said fimbrial adhesin is
selected from the group consisting of CfaE, CsfD, CsuD, CooD, CosD,
CsdD, CsbD and CotD.
14. The method of claim 10, wherein said major fimbrial subunit is
selected from the group consisting of CfaB, CsfA, CsuA1, CsuA2,
CooA, CosA, CsbA, CsdA and CotB.
15. The method of claim 10, wherein said immunogen contains a
polyhistidine tail linked at the C-terminus of said Escherichia
coli major fimbrial subunit.
16. The method of claim 13, wherein said fimbrial adhesin is the
amino acid sequence selected from the group consisting of SEQ ID
No. 1, SEQ ID No. 22, SEQ ID No. 27, SEQ ID No.28, SEQ ID No.29,
SEQ ID No.30, SEQ ID No.31, SEQ ID No.32 or fragments thereof.
17. The method of claim 13, wherein said CfaE is composed of the
amino acid sequence of SEQ ID No.11 encoded by all or a portion of
the nucleotide sequence of SEQ ID No. 18 or fragment thereof.
18. The method of claim 13, wherein said CsbD is composed of the
amino acid sequence of SEQ ID No.22 encoded by the nucleotide
sequence of SEQ ID No.19 or fragment thereof.
19. The method of claim 13, wherein said CotD is composed of the
amino acid sequence of SEQ ID No.32 or fragment thereof.
20. The method of claim 13, wherein said Escherichia coli fimbrial
adhesin is composed of amino acids 58-185 of the sequence selected
from the group consisting of SEQ ID No. 11, SEQ ID No.22, SEQ ID
No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ
ID No.32.
21. The method of claim 13, wherein said Escherichia coli fimbrial
adhesin is composed of amino acids 14-205 of the sequence selected
from the group consisting of SEQ ID No. 11, SEQ ID No.22, SEQ ID
No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ
ID No.32.
22. The method of claim 14, wherein said major fimbrial subunit is
the amino acid sequence selected from the group consisting of SEQ
ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID
No.6, SEQ ID No.7, SEQ ID No.8, and SEQ ID No.9.
23. The method of claim 15, wherein said immunogen is a fusion
polypeptide containing a polyhistidine tail composed of the amino
acid sequence selected from the group consisting of SEQ ID No. 23
encoded by SEQ ID No. 24, SEQ ID No. 25 encoded by SEQ ID No.26 and
SEQ ID No.34.
24. The method of claim 17, wherein said major fimbrial subunit is
CfaB with a polypeptide sequence of SEQ ID No. 1 encoded by
nucleotide sequence SEQ ID No. 20.
25. The method of claim 18, wherein said major fimbrial subunit is
CsbA with a polypeptide sequence of SEQ ID No. 7 encoded by
nucleotide sequence SEQ ID No. 21.
26. The method of claim 19, wherein said major fimbrial subunit is
CotA with a polypeptide sequence of SEQ ID. No. 9.
27. A method of conferring passive immunity to enterotoxigenic
Escherichia coli comprising: a. administering to a milk producing
animal an immunogen composed of native or recombinant Escherichia
coli adhesin so as to induce adhesin specific antibody in the
colostrums or milk of said milk producing animals; b. collecting
said colostrum or milk product containing said adhesin specific
antibody; c. administering a dose of said bovine milk anti-adhesin
immunoglobulin by ingestion wherein said dose is 0.1 g IgG per dose
to 20.0 g of IgG per dose.
28. The method of claim 27, wherein said dose is administered by
mixing said colostrum or milk anti-adhesin immunoglobulin in a
beverage or food.
29. The method of claim 27, wherein said dose is administered in
capsule or tablet form.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/683,787 filed May 24, 2005.
FIELD OF INVENTION
[0002] This inventive subject matter relates to a pharmaceutical
useful in conferring passive protection against diarrhea caused by
enterotoxigenic Escherichia coli.
SEQUENCE LISTING
[0003] I hereby state that the information recorded in computer
readable form is identical to the written sequence listing.
BACKGROUND OF INVENTION
[0004] Enterotoxigenic Escherichia coli (ETEC) are a principal
cause of diarrhea in young children in resource-limited countries
and also travelers to these areas (1, 2). ETEC produce disease by
adherence to small intestinal epithelial cells and expression of a
heat-labile (LT) and/or heat-stable (ST) enterotoxin (3). ETEC
typically attach to host cells via filamentous bacterial surface
structures known as colonization factors (CFs). More than 20
different CFs have been described, a minority of which have been
unequivocally incriminated in pathogenesis (4).
[0005] Firm evidence for a pathogenic role exists for colonization
factor antigen I (CFA/I), the first human-specific ETEC CF to be
described. CFA/I is the archetype of a family of eight ETEC
fimbriae that share genetic and biochemical features (5, 4, 6, 7).
This family includes coli surface antigen 1 (CS 1), CS2, CS4, CS
14, CS17, CS19 and putative colonization factor O71 (PCFO71). The
complete DNA sequences of the gene clusters encoding CFA/I, CS1 and
CS2 have been published (8, 9, 10, 11, 12). The genes for the major
subunit of two of the other related fimbriae have been reported
(13, 6). The four-gene bioassembly operons of CFA/I, CS1, and CS2
are similarly organized, encoding (in order) a periplasmic
chaperone, major fimbrial subunit, outer membrane usher protein,
and minor fimbrial subunit. CFA/I assembly takes place through the
alternate chaperone pathway, distinct from the classic
chaperone-usher pathway of type I fimbrial formation and that of
other filamentous structures such as type IV pili (14, 15). Based
on the primary sequence of the major fimbrial subunit, CFA/I and
related fimbriae have been grouped as class 5 fimbriae (16).
[0006] Studies of CS1 have yielded details on the composition and
functional features of Class 5 fimbriae (17). The CS1 fimbrial
stalk consists of repeating CooA major subunits. The CooD minor
subunit is allegedly localized to the fimbrial tip, comprises an
extremely small proportion of the fimbrial mass, and is required
for initiation of fimbrial formation (18). Contrary to earlier
evidence suggesting that the major subunit mediates binding (19),
recent findings have implicated the minor subunit as the adhesin
and identified specific amino acid residues required for in vitro
adhesion of CS1 and CFA/I fimbriae (20). The inferred primary amino
acid structure of those major subunits that have been sequenced
share extensive similarity. Serologic cross-reactivity of native
fimbriae is, however, limited, and the pattern of cross-reactivity
correlates with phylogenetically defined subtaxons of the major
subunits (13).
[0007] Implication of the minor subunits of Class 5 fimbriae as the
actual adhesins entreats scrutiny regarding the degree of their
conservation relative to that of the major subunits. It was
speculated that CooD and its homologs retained greater similarity
due to functional constraints imposed by ligand binding
requirements and/or its immunorecessiveness, itself attributable to
the extremely large ratio of major to minor subunits in terms of
fimbrial composition. Studies were conducted to examine the
evolutionary relationships of the minor and major subunits of Class
5 ETEC fimbriae as well as the two assembly proteins (21). It was
demonstrated that evolutionary distinctions exist between the Class
5 major and minor fimbrial subunits and that the minor subunits
function as adhesins. These findings provide practical implications
for vaccine-related research.
[0008] The nucleotide sequence of the gene clusters that encode
CS4, CS 14, CS17, CS19 and PCFO71 was determined from wild type
diarrhea-associated isolates of ETEC that tested positive for each
respective fimbria by monoclonal antibody-based detection (21). The
major subunit alleles of the newly sequenced CS4, CS14, CS17 and
CS19 gene clusters each showed 99-100% nucleotide sequence identity
with corresponding gene sequence(s) previously deposited in
GenBank, with no more than four nucleotide differences per allele.
Each locus had four open reading frames that encoded proteins with
homology to the CFA/I class chaperones, major subunits, ushers and
minor subunits. As previously reported (13), the one exception was
for the CS14 gene cluster, which contained two tandem open reading
frames downstream of the chaperone gene. Their predicted protein
sequences share 94% amino acid identity with one another and are
both homologous to other Class 5 fimbriae major subunits.
[0009] Examination of the inferred amino acid sequences of all the
protein homologs involved in Class 5 fimbrial biogenesis reveals
many basic similarities. Across genera, each set of homologs
generally share similar physicochemical properties in terms of
polypeptide length, mass, and theoretical isoelectric point. All of
the involved proteins contain an amino-terminal signal peptide that
facilitates translocation to the periplasm via the type II
secretion pathway. None of the major subunit proteins contain any
cysteine residues, while the number and location of six cysteine
residues are conserved for all of the minor subunits except that of
the Y. pestis homolog 3802, which contains only four of these six
residues.
[0010] Type 1 and P fimbriae have been useful models in elucidating
the genetic and structural details of fimbriae assembled by the
classical chaperone-usher pathway (23, 24, 25). An outcome of this
work has been development of the principle of donor strand
complementation, a process in which fimbrial subunits
non-covalently interlock with adjoining subunits by iterative
intersubunit sharing of a critical, missing .beta.-strand (22, 26).
Evidence has implicated this same mechanism in the folding and
quaternary conformational integrity of Haemophilus influenzae
hemagglutinating pili (28), and Yersinia pestis capsular protein, a
non-fimbrial protein polymer (29). Both of these structures are
distant Class I relatives of Type 1 and P fimbriae that are
assembled by the classical chaperone-usher pathway. From an
evolutionary perspective, this suggests that the mechanism of donor
strand complementation arose in a primordial fimbrial system from
which existing fimbriae of this Class have derived. While donor
strand complementation represents a clever biologic solution to the
problem of protein folding for noncovalently linked, polymeric
surface proteins, its exploitation by adhesive fimbriae other than
those of the classical usher-chaperone pathway has not been
demonstrated.
[0011] Common to fimbriae assembled by the alternate chaperone
pathway and the classical chaperone-usher pathway are the
requirement for a periplasmic chaperone to preclude subunit
misfolding and an usher protein that choreographs polymerization at
the outer membrane. That the fimbrial assembly and structural
components of these distinct pathways share no sequence similarity
indicates that they have arisen through convergent evolutionary
paths. Nevertheless, computational analyses of the CFA/I structural
subunits suggests the possibility that donor strand complementation
may also govern chaperone-subunit and subunit-subunit
interaction.
[0012] The eight ETEC Class 5 fimbriae clustered into three
subclasses of three (CFA/I, CS4, and CS14), four (CS1, PCFO71, CS17
and CS19), and one (CS2) member(s) (referred to as subclasses 5a,
5b, and 5c, respectively) (21). Previous reports demonstrated that
ETEC bearing CFA/I, CS2, CS4, CS14 and CS19 manifest adherence to
cultured Caco-2 cells (6, 22). However, conflicting data have been
published regarding which of the component subunits of CFA/I and
CS1 mediate adherence (19, 20).
[0013] This question of which fimbrial components is responsible
for mediating adherence was approached by assessing the
adherence-inhibition activity of antibodies to intact CFA/I
fimbriae, CfaB (major subunit), and to non-overlapping
amino-terminal (residues 23-211) and carboxy-terminal (residues
212-360) halves of CfaE (minor subunit) in two different in vitro
adherence models (21). It was demonstrated that the most important
domain for CFA/I adherence resides in the amino-terminal half of
the adhesin CfaE (21).
[0014] The studies briefly described above provide evidence that
the minor subunits of CFA/I and other Class 5 fimbriae are the
receptor binding moiety (20, 21). Consistent with these
observations, because of the low levels of sequence divergence of
the minor subunits observed within fimbrial subclasses 5a and 5b
(20), the evolutionary relationships correlated with
cross-reactivity of antibodies against the amino-terminal half of
minor subunits representing each of these two subclasses (21).
These studies strongly suggest that the minor subunits of class 5
fimbriae are much more effective in inducing antiadhesive immunity
and thus an important immunogen for inducing protective antibody
(21).
[0015] Anti-diarrheal vaccines would be a preferable method of
conferring protection against diarrheal disease including ETEC
caused diarrhea. However, because of the complexities of
constructing and licensing of effective vaccines other methods to
confer interim protection have been sought. A measure shown to hold
considerable promise in the prevention of diarrhea is passive, oral
administration of immunoglobulins against diarrhea causing
enteropathogens. Products with activity against ETEC, Shigella, and
rotavirus have been shown to prevent diarrhea in controlled studies
with anti-cryptosporidial bovine milk immunoglobulins (BIgG)
preparations (30-33). Furthermore, favorable encouraging results
have been observed using this approach with anti-cryptosporidial
BIgG preparations (34, 35).
[0016] Accordingly, an object of this invention is an
immunoglobulin supplement capable of providing prophylactic
protection against ETEC infection. Because the minor subunit
(adhesin) is the fimbrial component directly responsible for
adherence, administration of anti-adhesin antibodies will likely
confer significantly greater protection than antibodies raised
against intact fimbriae or intact bacteria. Furthermore, another
object of the invention is a method for the production of passive
prophylactic formulation against ETEC, containing anti-adhesin
immunoglobulin. The use of recombinant minor fimbrial subunit
polypeptides in the immunoglobulin production method will provide
enhanced antibody yields and standardization over the use of intact
fimbriae or whole cells.
SUMMARY OF INVENTION
[0017] Vaccines are the preferred method for conferring
anti-diarrhea protection in potentially exposed populations.
However, there are no currently licensed effective vaccines against
ETEC. Therefore, an interim protective measure, until vaccines can
be developed, is the administration of oral passive protection in
the form of anti-adhesin immunoglobulin supplements derived from
bovine, or other milk producing animal, colostrum or milk.
[0018] An object of the invention is a anti-Escherichia coli
antibody prophylactic formulation that is specific to class 5
enterotoxigenic E. coli fimbriae adhesin.
[0019] Another object of the invention is a method for conferring
passive immunity using an anti-E. coli antibody prophylactic
formulation that is specific to class five Escherichia coli
fimbriae adhesin including CfaE and CsbD.
[0020] An additional object of the invention is a method of
conferring passive immunity to enterotoxigenic E. coli by
administering a food supplement containing anti-E. coli antibody
specific to Class 5 fimbriae adhesins.
[0021] A still further object of the invention is a method of
producing an anti-E. coli adhesin milk antibody by administering
recombinant adhesin polypeptides to domestic animals such as
cows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. A highly conserved .beta.-strand motif in the major
structural subunits of Class 5 fimbriae. This is a multiple
alignment of the amino-termini of the mature form of the major
subunits, with consensus sequence shown below. This span is
predicted to form an interrupted .beta.-strand motif spanning
residues 5-19 (demarcated by yellow arrows below consensus).
Shading of conserved residues indicates class as follows: blue,
hydrophobic; red, negatively charged residues; turquoise,
positively charged residues; and green, proline. Also shown are the
sequence identification numbers (SEQ ID No.) for the associated
polypeptides. Abbrevations: Bcep, Burkholderia cepacia; Styp,
Salmonella typhi. U, hydrophobic residue; x, any residue; Z, E or
Q.
[0023] FIG. 2. Panel A, Schematic diagram showing the domains of
independent CfaE variant constructs with C-terminal extensions
comprising the N-terminal .beta.-strand span of CfaB varying in
length from 10 to 19 residues. Each construct contains a short
flexible linker peptide (DNKQ) intercalated between the C-terminus
of the native CfaE sequence and the donor .beta.-strand. The
vertical arrow identifies the donor strand valine that was modified
to either a proline (V7P) to disrupt the secondary .beta.-strand
motif. Panel B, Western blot analysis of periplasmic concentrates
from the series of strains engineered to express CfaE and the
variants complemented in cis with varying lengths of the
amino-terminal span of mature CfaB. The primary antibody
preparations used were polyclonal rabbit antibody against CfaE.
Lanes correspond to preparations from the following constructs:
Lane 1, dscloCfaE; 2, dsc11CfaE; 3, dsc12CfaE; 4, dsc13CfaE; 5,
dsc13CfaE[V7P]; 6, dsc14CfaE; 7, dsc16CfaE; 8, dsc19CfaE; and 9,
CfaE. Molecular weight markers (in kD) are shown to the left. Panel
C, schematic representation of the engineered components of dscl
9CfaE(His)6, containing the native CfaE sequence (including its
Sec-dependent N-terminal signal sequence), with an extension at its
C-terminus consisting of a short linker sequence (i.e., DNKQ), the
19 residue donor strand from the N-terminus of mature CfaB, and a
terminal hexahistidine affinity tag.
[0024] FIG. 3. Reactivity of products with a panel of CFA/I-related
antigens by ELISA.
[0025] FIG. 4. In vitro functional activity of antibodies to BIgG
anti-CfaE.
[0026] FIG. 5. Reactivity of products with a panel of CS17-related
antigens by ELISA.
[0027] FIG. 6. In vitro functional activity of BIgG anti-CsbD.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS
[0028] Vaccines are the preferred prophylactic measure for
long-term protection against ETEC caused diarrhea. However,
development of effective vaccines is typically difficult and
time-intensive. Furthermore, even after an effective ETEC vaccine
is developed, protection against ETEC caused disease is not
conferred until an adequate dose regimen is completed. Therefore,
there is a need for effective, safe and easy to take passive
prophylactic measures. A particularly promising approach, for
example, is the use of bovine milk immunoglobulins (BIgG)
preparations (30-33).
[0029] Computational analyses of the CFA/I structural subunits
suggest that donor strand complementation governs chaperone-subunit
and subunit-subunit interaction. The major subunits of Class 5
fimbriae share a highly conserved amino-terminal span predicted to
form a .beta. strand (FIG. 1). Based on its predicted structure and
location, the .beta.-strand-like structure is donated to
neighboring major subunit (e.g. CfaB) along the alpha-helical stalk
and to an adhesin (e.g. CfaE) at the fimbrial tip. The highly
conserved nature of the amino-terminal .beta. strand of CfaB and
its homologs, together with the precedent that the amino-terminus
of type 1 fimbrial subunits functions as the exchanged donor strand
in filament assembly suggested this as a good candidate for the
donor .beta. strand that noncovalently interlocks CFA/I
subunits.
[0030] ETEC fimbriae are classified based on genetic and structural
analysis and many fimbriae associated with disease fall into the
Class 5 fimbrial grouping, which includes CFA/I, CS17 and CS2.
Class 5 fimbriae adhesins each share significant characteristics
that clearly differentiate these members as belonging to a
recognizable genus. Although Class 5 fimbriae are distinguishable
serologically, they share similar architecture in that they are
composed of a major stalk forming subunit (e.g., CfaB of CFA/I) and
a minor tip-localized subunit (e.g., CfaE of CFA/I) that we have
found serves as the intestinal adhesin. A comparison of amino acid
sequences of the major and minor subunits (i.e., fimbrial adhesins)
clearly show a strong amino acid sequence relatedness as well as
sequence homology, as illustrated in Table 1, for the major
subunits and Table 2 for adhesin molecules. In Table 1 and 2 the
shaded areas show similarity of residues and the unshaded areas
show residue identity. As illustrated in Table 2, the fimbrial
adhesins display, as well as the major subunits, a high level of
residue identity, ranging from 47% to 98%. Additionally, fimbrial
adhesins have significant amino acid sequence conservation,
including a conserved structural motif in the carboxy-terminal
domain of both the major and minor subunits (i.e., the beta-zipper
motif). This structure indicates that the C-terminal domain of
these proteins are involved in subunit-subunit interaction.
TABLE-US-00001 TABLE 1 CfaB CooA CotA CsfA CsuA.sub.1 CsbA CsdA
CosA CfaB -- .53 .51 .66 .58 .50 .52 .52 CooA .74 -- .50 .57 .51
.61 .59 .90 CotA .67 .71 -- .50 .52 .45 .47 .52 CsfA .81 .75 .71 --
.62 .54 .55 .56 CsuA.sub.1 .75 .72 .71 .78 -- .51 .50 .52 CsbA .73
.76 .67 .72 .73 -- .88 .61 CsdA .72 .75 .69 .71 .72 .92 -- .57 CosA
.71 .95 .74 .73 .73 .79 .78 -- 3 letter codes: CFA/I, Cfa; CS1,
Coo; CS2, Cot; CS4, Csf; CS14, Csu; CS17, Csb; CS19, Csd; PCFO71,
Cos.
[0031] TABLE-US-00002 TABLE 2 CfaE CooD CotD CsfD CsuD CsbD CsdD
CosD CfaE -- .51 .46 .80 .82 .51 .51 .50 CooD .65 -- .49 .51 .50
.97 .97 .98 CotD .64 .63 -- .47 .48 .48 .48 .48 CsfD .87 .65 .64 --
.94 .50 .50 .50 CsuD .88 .64 .65 .97 -- .50 .50 .50 CsbD .66 .97
.62 .65 .65 -- .97 .96 CsdD .66 .97 .63 .64 .65 .97 -- .98 CosD .65
.99 .62 .64 .65 .96 .97 -- 3 letter codes: CFA/I, Cfa; CS1, Coo;
CS2, Cot; CS4, Csf; CS14, Csu; CS17, Csb; CS19, Csd; PCFO71,
Cos.
[0032] Toward the development of an ETEC antigen, we constructed a
conformationally-stable construct wherein an amino-terminal donor
.beta.-strand of CfaB provides an in cis carboxy-terminal extension
of CfaE to confer conformational stability and protease resistance
to this molecule, forming a soluble monomer capable of binding
human erythrocytes. In order to identify common structural motifs,
multiple alignments of the amino acid sequences of the eight
homologs of the major and minor subunits of Class 5 ETEC fimbriae
were generated. Secondary structure prediction algorithms indicated
that both subunits form an amphipathic structure rich in
.beta.-strands distributed along their length. Twenty six percent
of the consensus minor subunit sequence is predicted to fold into a
.beta.-conformation, comprising 17 interspersed .beta. strands,
which might be expected to form a hydrophobic core. Sakellaris et
al have previously suggested that an amino acid span forms a
.beta.-zipper motif, analogous to that of class I fimbrial
subunits, that plays a role in fimbrial subunit-chaperone
interaction (27).
[0033] The following example discloses the production of a CfaE
immunogen using a donor strand from CfaB. However, one of skill in
the art, following this disclosure, would be able to engineer
constructs to serve as an immunogen using donor strands from other
class 5 major subunits in conjunction with other adhesin
constructs, such as CsbD, CsfD, CsuD, CooD, CosD, CsdD, and CotD.
The major Class 5 fimbrial subunits are listed in Table 3 along
with the corresponding SEQ ID No. corresponding to the subunit's
amino acid sequence donor strand. Table 4 lists the amino acid
sequence of the Class 5 adhesin and their respective SEQ ID No.
TABLE-US-00003 TABLE 3 SEQ ID No. of Donor Strand Major Subunit
Amino Acid Sequence CfaB 1 CsfA 2 CsuA1 3 CsuA2 4 CooA 5 CosA 6
CsbA 7 CsdA 8 CotB 9
[0034] TABLE-US-00004 TABLE 4 Minor Subunit SEQ ID. No. of Amino
Acid Sequence CfaE 11 CsbD 22 CsfD 27 CsuD 28 CooD 29 CosD 30 CsdD
31 CotD 32
[0035] An inventive aspect of this invention is a method for the
production of a passive prophylactic against Class 5 fimbrial
adhesin of ETEC bacteria. Examples using specific Class 5 fimbrial
adhesins are provided in order to illustrate the invention.
However, other Class 5 fimbrial adhesins, and their associated
major subunits can also be utilized by one of skill in the art
EXAMPLE 1
Production of anti-CfaE Bovine Immunoglobulin
[0036] As mentioned above, the highly conserved nature of the
amino-terminal .beta. strand of CfaB and its homologs, together
with other structure/function studies in type 1 fimbrial subunits,
suggested this structure as a good candidate for the donor .beta.
strand that interlocks CFA/I subunits. In order to test this
hypothesis with respect to the minoradhesive subunit, a plasmid was
engineered to express a CfaE variant containing a C-terminal
extension consisting of a flexible hairpin linker (DNKQ, SEQ ID No.
10) followed by an amino acid sequence of CfaB (FIG. 2). It was
found that a CfaB donor strand length of at least 12 to as many as
19 amino acids was necessary to obtain a measurable recovery of
CfaE. In studies using constructs containing a 12 to 19 amino acid
donor strand, where mutations were introduced to break the .beta.
strand, it was demonstrated that the .beta. strand is important to
the observed stability achieved by the C-terminal amino acid
extension. It was further determined that the C-terminal .beta.
strand contributed by CfaB in cis precludes chaperone (e.g.
CfaA)-adhesin complex formation.
[0037] In this example, a recombinant CfaE antigen was constructed,
as shown in FIG. 2C, by fusing a Cfa E polypeptide sequence (SEQ ID
No. 11), encoded by the nucleotide sequence of SEQ ID No. 18 to the
N-terminal amino acid sequence of a linker polypeptide (SEQ ID No.
10) which is in-turn linked at its C-terminus to a 19 amino acid
CfaB donor strand corresponding to amino acids 1-19 of SEQ ID No.
1. Although, SEQ ID No. 10 was utilized for a linker, other amino
acid sequences have been found acceptable, including SEQ ID No. 12
and 13. For this example, the CfaB major subunit donor strand used
is shown in SEQ ID No. 1 which is encoded by the nucleotide
sequence of SEQ ID No. 20. However, based on the observation that
the a donor strand of 12 to 19 amino acids is suitable for
significant CfaE recovery, a recombinant antigen containing 12 to
19 amino acids can be utilized. Similarly, recombinant peptides can
be constructed containing all or a portion of SEQ ID No. 11 as long
as the amino acid sequence contains anti-CfaE B-cell epitopes.
[0038] The CfaE construct containing the 19 amino acid major
subunit donor strand was constructed by first inserting cfaE into
plasmid vectors by in vitro recombination using the Gateway.RTM.
system (Invitrogen, Carlsbad, Calif.). Primers with the following
sequences were used for the initial cloning into pDONR20.TM.:
dsc-CfaE 13-1 (forward), 5'-TCG ACA ATA AAC AAG TAG AGA AAA ATA TTA
CTG TAA CAG CTA GTG TTG ATC CTT AGC-3' (SEQ ID No. 14); and
dsc-CfaE 13-2 (reverse), 5'-TCG AGC TAA GGA TCA ACA CTA GCT GTT ACA
GTA ATA TTT TTC TCT ACT TGT TTA TTG-3' (SEQ ID No 15). The PCR
products flanked by attB recombination sites were cloned into the
donor vector pDONR201 (Gateway.RTM. Technology, Invitrogen,
Carlsbad, Calif.), using the Gateway BP.RTM. reaction to generate
the entry vector pRA13.3. In the Gateway LR.RTM. reaction the gene
sequence was further subcloned from pRA13.3 into the modified
expression vector pDEST14-Kn.sup.r (vector for native expression
from a T7 promoter) to generate the plasmid pRA14.2. The
pDEST14-Kn.sup.r vector was constructed by modifying pDEST14.RTM.
(Gateway.RTM. Technology, Invitrogen, Carlsbad, Calif.) by
replacement of ampicillin with kanamycin resistance. The presence
of the correct cfaE was confirmed by sequence analysis. E. coli
strain BL21 SI (Invitrogen, Carlsbad, Calif.) was used for the
expression of the pRA14.1 and related CfaE donor strand
complemented constructs.
[0039] The above procedure was utilized to construct a CfaE/donor
strand recombinant construct. However, constructs containing other
adhesin molecules can also be constructed, including the minor
subunits: CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD, in
conjunction with the appropriate donor strand from the major
subunits as listed in Table 1. For example, a recombinant CsbD
construct was designed comprising a CsbD polypeptide sequence
comprising all or a portion of SEQ ID No. 22 fused at the
C-terminal end, via a linker polypeptide of SEQ ID No 10, to a CsbA
major subunit donor strand of a polypeptide sequence SEQ ID No. 6
that is encoded by the nucleotide sequence of SEQ ID No. 21.
Development of pET/Adhesin Construct for Large Scale Antigen
Production
[0040] The DNA construct encoding dsclgCfaE was then excised from
pDEST14.RTM. vector and inserted into pET24(a).TM. in order to
encode a variant CfaE construct that incorporates a
carboxy-terminal polyhistine tail after the CfaB donor strand. This
construct, with a polypeptide sequence of SEQ ID No 23 is
designated dsc.sub.19CfaE(His).sub.6 and is encoded by the
nucleotide sequence of SEQ ID No. 24.
[0041] Construction of the dsc.sub.19cfaE insert was carried out by
amplifying the pDEST 14 vector by polymerase chain reaction using a
NdeI containing forward primer and an XhoI containing reverse
primer, SEQ ID No 16 and 17, respectively. The dsc.sub.19cfaE
coding region was directionally ligated into an NdeI/XhoI
restricted pET24a plasmid. The insert containing pET24a.TM. plasmid
was used to transform NovaBlue-3.TM. BL21 (EMD Biosciences,
Novagen.RTM. Brand, Madison, Wis.) bacteria. Transformed colonies
were then selected and re-cultured in order to expand the plasmid
containing bacteria. Plasmid inserts from selected colonies were
then sequenced. These plasmids were then re-inserted into BL21
(DE3) (EMD Biosciences, Novagen.RTM. Brand, Madison, Wis.)
competent cells and the DNA insert sequence confirmed.
[0042] Similar to the method used to construct
dsc.sub.19CfaE(His).sub.6, a DNA construct encoding dsc.sub.19CsbD
was also made by insertion of CsbD and a CsbA donor strain sequence
into pET24a.TM.. This construct has a polypeptide sequence of SEQ
ID No. 25 and is encoded by the nucleotide sequence of SEQ ID No.
26. The donor strand sequence from CsbA used in designing the
construct is disclosed as SEQ ID No. 6. Like the CfaE construct,
the 19 amino acid sequence from CsbA corresponding to amino acids
1-19 of SEQ ID No. 6 was used. However donor strand sequences
ranging from the 12 to 19 amino acids can be used.
Production of dsc.sub.19CfaE(His).sub.6.
[0043] A number of growth conditions and media can be utilized for
large-scale production of the dsc.sub.19CfaE(His).sub.6, or other
adhesin/donor strand construct. For example initiation of culture
can be conducted using 1.0 .mu.M to 1.0 mM
isopropyl-.beta.-D-thiogalactopyranosid (IPTG) at an induction
temperature of 320 C to 25.degree. C. for 1 to 4 hours. In this
example, LB media was utilized with a 1.0 .mu.M IPTG concentration
at 32.degree. C. for 3 hours. However, APS.TM. and other media
formulations can also be used. The dsc.sub.19CfaE(His).sub.6, or
other recombinant adhesin construct, is purified on a Ni column.
Yield of construct is at least 0.45 to 0.9 mg of protein/L of
culture.
Manufacture of BIgG
[0044] Antibody to recombinant antigen is produced in the colostrum
or milk of domesticated cattle, including Holsteins. A total of
three intramuscular vaccinations each in a volume of two ml
containing 500 .mu.g of antigen each is administered at a single
site. Vaccinations are given approximately three weeks apart with
the final vaccination 1 to 2 weeks prior to calving. At calving the
first four milkings are collected, the volume estimated and a
sample tested for anti-adhesin antibody by enzyme-linked
immunosorbent assay (ELISA). FIG. 3 shows the reactivity of
anti-CFA/I BIgG and anti-CfaE BIgG products. CFA/I BIgG gives a
higher level of reactivity to CFA/I antigen than anti-CfaE by ELISA
(FIG. 3A). This is due to the fact that CFA/I antigen used to coat
the ELISA plate is made of primarily the CfaB major subunit and the
CfaE minor subunit is present as a minor component only. As
expected, the anti-CfaE BIgG product has a much stronger reaction
with CfaE compared to either AEMI or anti-CFA/I BIgG (FIG. 3B).
This confirms that immunization of cows with the CfaE antigen
greatly enhances the generation of antibodies to adhesin, CfaE.
[0045] Further processing of the collected product can be
undertaken. For example, frozen milk is fractionated to remove
caseins through a cheese-making step. The whey fraction, containing
most immunoglobulins is then drained from the cheese curd and
pasteurized under standard dairy conditions. The
immunoglobulin-enriched whey fraction is then concentrated and
residual milk fat is removed by centrifugation at room temperature.
Subsequently, phospholipid and non-immunoglobulin proteins can be
removed (36). The final product is then concentrated to 15-20%
solids and salts removed by continuous diafiltration against three
buffer changes. The final product is then tested for by ELISA.
[0046] In addition to the characterization of antibody reactivity
of BIgG to ETEC antigens, the functional activity of the antibodies
was evaluated. As the receptor(s) for CFA/I is not defined, a
surrogate assay for adhesion of ETEC to target cells in vitro was
used. ETEC expressing certain fimbriae (including CFA/I) adhere to
and agglutinate human and/or bovine erythrocytes in a
mannose-resistant hemagglutination assay (MRHA). This is used as a
surrogate marker for adhesion of ETEC whole cells, fimbriae or
purified minor subunits of fimbriae to target eukaryotic epithelial
cells. This phenomenon, described as hemagglutination inhibition
(HAI), is an indicator of antibodies capable of neutralizing
adhesion of ETEC to target cells.
[0047] In FIG. 4, human erythrocytes were agglutinated by ETEC
expressing CFA/I, CS4 or CS14 in a mannose-resistant manner (MRHA).
This MRHA can be inhibited by pre-incubation of bacteria with
anti-CFA/I BIgG or anti-CfaE BIgG. Shown in FIG. 4, both anti-CFA/I
BIgG and anti-CfaE BIgG contained antibodies capable of inhibiting
the ability of ETEC that express the homologous fimbriae from
agglutinating human erythrocytes. FIG. 4 shows the titer of BIgG
(expressed as mg IgG/ml) required to neutralized aggluntination of
bovine erythrocytes by ETEC expressing different colonization
factors. The concentrations of BIgG products tested were adjusted
so the minimal concentrations of IgG were equal in both products.
Therefore, the data is expressed as the concentration of IgG that
is required to inhibit MRHA by ETEC expressing CFA/I, CS4 or CS14
fimbriae. As little as 14 to 17 .mu.g/ml of bovine IgG present in
the BIgG powders are required in vitro to inhibit MRHA.
[0048] Strong inhibitory activity is provided by anti-CFA/I, as
expected, with an equivalent level of inhibition provide by
anti-CfaE. Of importance is that both anti-CFA/I and anti-CfaE show
cross-reactivity of binding inhibition against CS4 and CS14. This
illustrates that an anti-CfaE prophylactic antibody will have
utility in conferring protection against other related
antigens.
EXAMPLE 2
Production of Anti-CfaD (CS17) Bovine Immunoglobulin
[0049] Use of other class 5 fimbrial adhesins are also contemplated
as eliciting protective passive antibody production. As a further
illustration, results of inhibition by antibody to CS17 (i.e.,
CsbD) is presented in FIG. 5. The antigen used to elicit antibody
was a CsbD polypeptide (SEQ ID No. 22) expressing construct. The
construct was engineered similar to that for CfaE, in Example 1,
above but with a nucleotide sequence encoding CsbD (SEQ ID No. 19).
The construct was designated dsc.sub.19CsbD[His].sub.6. The donor
strand consisted of 19 amino acids of CsbA (SEQ ID No. 7).
[0050] As can be seen in FIG. 5, like that for CfaE, antibody to
CsbD was highly efficient at inhibiting MRHA. Also, like that
observed for CfaE, anti-CsbD antibody also afforded
cross-protection against CS4 and CS2.
[0051] The functional activity of BIgG to CS17 and CsbD was also
evaluated, as in FIG. 4. These results are illustrated in FIG. 6.
Like that observed for anti-CfaE and anti-CFA/I, BIgG against both
CS17 and CsbD exhibited significant inhibitory activity. However,
more pronounce than for anti-CfaE BIgG, anti-CsbD, compared to
anti-CS17 BIgG, exhibited significant inhibitory activity even to
heterologous antigens. These observations, along with that observed
for CfaE indicate that only a limited number of species within the
Class five adhesin genus is likely to be required for efficacious
passive protection.
EXAMPLE 3
Specific Regions of ETEC Fimbrial Adhesin are Important for
Immunoreactivity and Stability
[0052] Crystollgraphic analysis of the dscCfaE reveals that
fimbrial adhesin is composed of two domains, an adhesin domain,
formed by the amino-terminal segment of the adhesin molecule and a
C-terminal pilin domain. The two domains are separated by a three
amino acid linker. In an attempt to understand those regions of
fimbrial adhesin, amino acid substitutions where made and the
ensuing immunoreactivity analyzed. It was found that replacement of
arginine 67 or arginine 181 with alanine, on CfaE abolishes the in
vitro adherence phenotype of the molecule. These amino acids
positions are located on exposed regions of the molecule with
residue Arg 181 located on the distal portion of the amino-terminus
of the domain. Therefore, this region of CfaE and the comparable
region of the other fimbrial adhesins, is important for efficacious
immune induction. Table 3 summarizes the positions in the eight
adhesins. Also shown in Table 3 is that region of the domain that
has added importance, based on crystollgraphic analysis, in
conferring structural stability of the fimbrial adhesin molecule.
TABLE-US-00005 TABLE 3 Fimbrial Adhesin domain Fimbrial Adhesin
domain residues important for residues important for Fimbrial
Adhesin immunoreactivity structural stability CfaE amino acids
66-183 amino acids 22-202 CsuD amino acids 66-183 amino acids
22-202 CsfD amino acids 66-183 amino acids 22-202 CooD amino acids
65-183 amino acids 20-205 CosD amino acids 65-185 amino acids
20-205 CsbD amino acids 65-183 amino acids 20-205 CsdB amino acids
65-183 amino acids 20-205 CotD amino acids 58-177 amino acids
14-196
[0053] Stabilization of the adhesin domain of intact fimbrial
adhesin molecules is provided by the major subunit. However, devoid
of the pili domain, fimbrial adhesin exhibits greater
conformational stability than the intact molecule with concomitant
retention of immunoreactivity. As an alternative to administration
of the intact adhesin molecule, administration of only the adhesin
domain is an alternative immunogen for induction of anti-fimbrial
adhesin antibodies. Therefore, as an example, recombinant adhesin
domain constructs encoding CfaE, CsbD and CotD adhesin domains, but
not containing the pili domain, were constructed, by polymerase
chain reaction amplification of the adhesin domain and inserted
into pET 24a.TM.. The amino acid sequences of the recombinant
product is illustrated in SEQ ID No.s 35, 36 and 37. Incorporation
of a polyhistidine tail, as in Example 1 and 2, facilitates
purification of the ensuing expressed product.
EXAMPLE 4
Administration of Anti-Fimbrial Adhesin as Prophylactic against
ETEC
[0054] Class five fimbrial adhesins can be used for the development
of prophylactic protection against ETEC infection. Protection is
provided by collecting colostrums or milk product from fimbrial
adhesin, either native or recombinant Escherichia coli adhesin,
immunizing cows. Immunization can be by any number of methods.
However, a best mode is the administration of three doses
intramuscularly three weeks apart with a final administration, 1 to
2 weeks prior to calving, of se in 1 to 2 ml volume containing up
to 500 .mu.g of said adhesin. Collection of milk or colostrums can
be at anytime, however optimal results likely is when collection is
1 to 2 weeks prior to calving.
[0055] Administration of the anti-adhesin bovine immunoglobulin as
a prophylactic is achieved by ingestion of 0.1 g IgG/dose to 20.0 g
of IgG/dose. The anti-adhesin bovine colostrum or milk
immunoglobulin can be ingested alone or mixed with a number of
beverages or foods, such as in candy. The immunglobulin can also be
reduced to tablet or capusular form and ingested.
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Sequence CWU 1
1
32 1 20 PRT Escherichia coli 1 Val Glu Lys Asn Ile Thr Val Thr Ala
Ser Val Asp Pro Val Ile Asp 1 5 10 15 Leu Leu Gln Ala 20 2 20 PRT
Escherichia coli 2 Val Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp
Pro Thr Ile Asp 1 5 10 15 Ile Leu Gln Ala 20 3 20 PRT Escherichia
coli 3 Val Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp Pro Thr Ile
Asp 1 5 10 15 Ile Leu Gln Ala 20 4 20 PRT Escherichia coli 4 Val
Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp Pro Thr Ile Asp 1 5 10
15 Ile Leu Gln Ala 20 5 20 PRT Escherichia coli 5 Val Glu Lys Thr
Ile Ser Val Thr Ala Ser Val Asp Pro Thr Val Asp 1 5 10 15 Leu Leu
Gln Ser 20 6 20 PRT Escherichia coli 6 Val Glu Lys Thr Ile Ser Val
Thr Ala Ser Val Asp Pro Thr Val Asp 1 5 10 15 Leu Leu Gln Ser 20 7
20 PRT Escherichia coli 7 Val Glu Lys Asn Ile Thr Val Arg Ala Ser
Val Asp Pro Lys Leu Asp 1 5 10 15 Leu Leu Gln Ala 20 8 20 PRT
Escherichia coli 8 Val Glu Lys Asn Ile Thr Val Arg Ala Ser Val Asp
Pro Lys Leu Asp 1 5 10 15 Leu Leu Gln Ala 20 9 20 PRT Escherichia
coli 9 Ala Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp Pro Thr Ile
Asp 1 5 10 15 Leu Met Gln Ser 20 10 4 PRT Escherichia coli 10 Asp
Asn Lys Gln 1 11 360 PRT Escherichia coli 11 Met Asn Lys Ile Leu
Phe Ile Phe Thr Leu Phe Phe Ser Ser Gly Phe 1 5 10 15 Phe Thr Phe
Ala Val Ser Ala Asp Lys Asn Pro Gly Ser Glu Asn Met 20 25 30 Thr
Asn Thr Ile Gly Pro His Asp Arg Gly Gly Ser Ser Pro Ile Tyr 35 40
45 Asn Ile Leu Asn Ser Tyr Leu Thr Ala Tyr Asn Gly Ser His His Leu
50 55 60 Tyr Asp Arg Met Ser Phe Leu Cys Leu Ser Ser Gln Asn Thr
Leu Asn 65 70 75 80 Gly Ala Cys Pro Ser Ser Asp Ala Pro Gly Thr Ala
Thr Ile Asp Gly 85 90 95 Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu
Lys Arg Ser Leu Ile Lys 100 105 110 Arg Glu Leu Gln Ile Lys Gly Tyr
Lys Gln Phe Leu Phe Lys Asn Ala 115 120 125 Asn Cys Pro Ser Lys Leu
Ala Leu Asn Ser Ser His Phe Gln Cys Asn 130 135 140 Arg Glu Gln Ala
Ser Gly Ala Thr Leu Ser Leu Tyr Ile Pro Ala Gly 145 150 155 160 Glu
Leu Asn Lys Leu Pro Phe Gly Gly Val Trp Asn Ala Val Leu Lys 165 170
175 Leu Asn Val Lys Arg Arg Tyr Asp Thr Thr Tyr Gly Thr Tyr Thr Ile
180 185 190 Asn Ile Thr Val Asn Leu Thr Asp Lys Gly Asn Ile Gln Ile
Trp Leu 195 200 205 Pro Gln Phe Lys Ser Asn Ala Arg Val Asp Leu Asn
Leu Arg Pro Thr 210 215 220 Gly Gly Gly Thr Tyr Ile Gly Arg Asn Ser
Val Asp Met Cys Phe Tyr 225 230 235 240 Asp Gly Tyr Ser Thr Asn Ser
Ser Ser Leu Glu Ile Arg Phe Gln Asp 245 250 255 Asp Asn Ser Lys Ser
Asp Gly Lys Phe Tyr Leu Lys Lys Ile Asn Asp 260 265 270 Asp Ser Lys
Glu Leu Val Tyr Thr Leu Ser Leu Leu Leu Ala Gly Lys 275 280 285 Asn
Leu Thr Pro Thr Asn Gly Gln Ala Leu Asn Ile Asn Thr Ala Ser 290 295
300 Leu Glu Thr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu Ile
305 310 315 320 Ser Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu
Asp Ala Lys 325 330 335 Val Lys Asn Pro Glu Ala Gly Gln Tyr Met Gly
Asn Ile Lys Ile Thr 340 345 350 Phe Thr Pro Ser Ser Gln Thr Leu 355
360 12 5 PRT Escherichia coli 12 Gly Asp Asn Lys Gln 1 5 13 6 PRT
Escherichia coli 13 Gly Asp Asn Lys Gln Gly 1 5 14 57 DNA
Escherichia coli 14 tcgacaataa acaagtagag aaaaatatta ctgtaacagc
tagtgttgat ccttagc 57 15 57 DNA Escherichia coli 15 tcgagctaag
gatcaacact agctgttaca gtaatatttt tctctacttg tttattg 57 16 45 DNA
Escherichia coli 16 cgcggggaat tccatatgaa taaaatttta tttattttta
cattg 45 17 36 DNA Escherichia coli 17 cgcccgctcg agttgcaaaa
gatcaatcac aggatc 36 18 1080 DNA Escherichia coli 18 atgaataaaa
ttttatttat ttttacattg tttttttctt cagggttttt tacatttgcc 60
gtatcggcag ataaaaatcc cggaagtgaa aacatgacta atactattgg tccccatgac
120 agggggggat cttcccccat atataatatc ttaaattcct atcttacagc
atacaatgga 180 agccatcatc tgtatgatag gatgagtttt ttatgtttgt
cttctcaaaa tacactgaat 240 ggagcatgcc caagcagtga tgcccctggc
actgctacaa ttgatggcga aacaaatata 300 acattacaat ttacggaaaa
aagaagtcta attaaaagag aactgcaaat taaaggctat 360 aaacaatttt
tgttcaaaaa tgctaattgc ccatctaaac tagcacttaa ctcatctcat 420
tttcaatgta atagagaaca agcttcaggt gctactttat cgttatacat accagctggt
480 gaattaaata aattaccttt tgggggggtc tggaatgccg ttctgaagct
aaatgtaaaa 540 agacgatatg atacaaccta tgggacttac actataaaca
tcacagttaa tttaactgat 600 aagggaaata ttcagatatg gttaccacag
ttcaaaagta acgctcgtgt cgatcttaac 660 ttgcgtccaa ctggtggtgg
tacatatatc ggaagaaatt ctgttgatat gtgcttttat 720 gatggatata
gtactaacag cagctcttta gagataagat ttcaggatga taattctaaa 780
tctgatggaa aattttatct aaagaaaata aatgatgact ccaaagaact tgtatacact
840 ttgtcacttc tcctggcagg taaaaattta acaccaacaa atggacaggc
attaaatatt 900 aacactgctt ctctggaaac aaactggaat agaattacag
ctgtcaccat gccagaaatc 960 agtgttccgg tgttgtgttg gcctggacgt
ttgcaattgg atgcaaaagt gaaaaatccc 1020 gaggctggac aatatatggg
gaatattaaa attactttca caccaagtag tcaaacactc 1080 19 1089 DNA
Escherichia coli 19 atgaaaaaga tatttatttt tttgtctatc atattttctg
cggtggtcag tgccgggcga 60 tacccggaaa ctacagtagg taatctgacg
aagagttttc aagcccctcg tcaggataga 120 agcgtacaat caccaatata
taacatcttt acgaatcatg tggctggata tagtttgagt 180 cataacttat
atgacaggat tgttttttta tgtacatcct cgtcgaatcc ggttaatggt 240
gcttgcccaa cccttggaac atctggagtt caatacggta ctacaaccat aaccttgcag
300 tttacagaaa aaagaagtct gataaaaaga aatattaatc ttgcaggtaa
taagaaacca 360 atatgggaga atcagagttg cgacactagc aatctaatgg
tgttgaattc gaagtcttgg 420 tcctgtgggc attacggaaa tgctaacgga
acacttctaa atctgtatat ccctgcagga 480 gaaatcaaca aattgccttt
tggagggata tgggaggcaa ctctgatctt acgcttatca 540 agatatggcg
aagtcagtag cacccattac ggcaattata ccgtaaatat tacggttgat 600
ttaactgata aaggtaatat tcaggtatgg cttccagggt ttcacagcaa cccgcgtgta
660 gacctgaatc tgcaccctat cggtaattat aaatatagtg gtagtaattc
actcgacatg 720 tgtttctatg atggatatag tacaaacagt gatagcatgg
taataaagtt ccaggatgat 780 aatcctacct attcatctga atataatctt
tataagatag ggggcactga aaaattaccc 840 tatgctgttt cactgcttat
gggagaaaaa atattttatc cagtgaatgg tcaatcattt 900 actatcaatg
acagtagtgt actcgaaaca aactggaatc gagtaaccgc agttgctatg 960
ccggaagtta atgttccagt attatgctgg ccagcaagat tgctattaaa tgctgatgta
1020 aatgctcccg atgcaggaca gtattcagga cagatatata taacatttac
acccagtgtc 1080 gaaaattta 1089 20 63 DNA Escherichia coli 20
gtagagaaaa atattactgt aacagctagt gttgatcctg tgattgatct tttgcaactc
60 gag 63 21 63 DNA Escherichia coli 21 gtcgaaaaaa atattactgt
gagggcaagt gttgacccta aacttgatct tctgcaactc 60 gag 63 22 363 PRT
Escherichia coli 22 Met Lys Lys Ile Phe Ile Phe Leu Ser Ile Ile Phe
Ser Ala Val Val 1 5 10 15 Ser Ala Gly Arg Tyr Pro Glu Thr Thr Val
Gly Asn Leu Thr Lys Ser 20 25 30 Phe Gln Ala Pro Arg Gln Asp Arg
Ser Val Gln Ser Pro Ile Tyr Asn 35 40 45 Ile Phe Thr Asn His Val
Ala Gly Tyr Ser Leu Ser His Asn Leu Tyr 50 55 60 Asp Arg Ile Val
Phe Leu Cys Thr Ser Ser Ser Asn Pro Val Asn Gly 65 70 75 80 Ala Cys
Pro Thr Leu Gly Thr Ser Gly Val Gln Tyr Gly Thr Thr Thr 85 90 95
Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys Arg Asn Ile 100
105 110 Asn Leu Ala Gly Asn Lys Lys Pro Ile Trp Glu Asn Gln Ser Cys
Asp 115 120 125 Thr Ser Asn Leu Met Val Leu Asn Ser Lys Ser Trp Ser
Cys Gly His 130 135 140 Tyr Gly Asn Ala Asn Gly Thr Leu Leu Asn Leu
Tyr Ile Pro Ala Gly 145 150 155 160 Glu Ile Asn Lys Leu Pro Phe Gly
Gly Ile Trp Glu Ala Thr Leu Ile 165 170 175 Leu Arg Leu Ser Arg Tyr
Gly Glu Val Ser Ser Thr His Tyr Gly Asn 180 185 190 Tyr Thr Val Asn
Ile Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln 195 200 205 Val Trp
Leu Pro Gly Phe His Ser Asn Pro Arg Val Asp Leu Asn Leu 210 215 220
His Pro Ile Gly Asn Tyr Lys Tyr Ser Gly Ser Asn Ser Leu Asp Met 225
230 235 240 Cys Phe Tyr Asp Gly Tyr Ser Thr Asn Ser Asp Ser Met Val
Ile Lys 245 250 255 Phe Gln Asp Asp Asn Pro Thr Tyr Ser Ser Glu Tyr
Asn Leu Tyr Lys 260 265 270 Ile Gly Gly Thr Glu Lys Leu Pro Tyr Ala
Val Ser Leu Leu Met Gly 275 280 285 Glu Lys Ile Phe Tyr Pro Val Asn
Gly Gln Ser Phe Thr Ile Asn Asp 290 295 300 Ser Ser Val Leu Glu Thr
Asn Trp Asn Arg Val Thr Ala Val Ala Met 305 310 315 320 Pro Glu Val
Asn Val Pro Val Leu Cys Trp Pro Ala Arg Leu Leu Leu 325 330 335 Asn
Ala Asp Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile 340 345
350 Tyr Ile Thr Phe Thr Pro Ser Val Glu Asn Leu 355 360 23 1176 DNA
Escherichia coli 23 atgaataaaa ttttatttat ttttacattg tttttttctt
cagggttttt tacatttgcc 60 gtatcggcag ataaaaatcc cggaagtgaa
aacatgacta atactattgg tccccatgac 120 agggggggat cttcccccat
atataatatc ttaaattcct atcttacagc atacaatgga 180 agccatcatc
tgtatgatag gatgagtttt ttatgtttgt cttctcaaaa tacactgaat 240
ggagcatgcc caagcagtga tgcccctggc actgctacaa ttgatggcga aacaaatata
300 acattacaat ttacggaaaa aagaagtcta attaaaagag aactgcaaat
taaaggctat 360 aaacaatttt tgttcaaaaa tgctaattgc ccatctaaac
tagcacttaa ctcatctcat 420 tttcaatgta atagagaaca agcttcaggt
gctactttat cgttatacat accagctggt 480 gaattaaata aattaccttt
tgggggggtc tggaatgccg ttctgaagct aaatgtaaaa 540 agacgatatg
atacaaccta tgggacttac actataaaca tcacagttaa tttaactgat 600
aagggaaata ttcagatatg gttaccacag ttcaaaagta acgctcgtgt cgatcttaac
660 ttgcgtccaa ctggtggtgg tacatatatc ggaagaaatt ctgttgatat
gtgcttttat 720 gatggatata gtactaacag cagctcttta gagataagat
ttcaggatga taattctaaa 780 tctgatggaa aattttatct aaagaaaata
aatgatgact ccaaagaact tgtatacact 840 ttgtcacttc tcctggcagg
taaaaattta acaccaacaa atggacaggc attaaatatt 900 aacactgctt
ctctggaaac aaactggaat agaattacag ctgtcaccat gccagaaatc 960
agtgttccgg tgttgtgttg gcctggacgt ttgcaattgg atgcaaaagt gaaaaatccc
1020 gaggctggac aatatatggg gaatattaaa attactttca caccaagtag
tcaaacactc 1080 gacaataaac aagtagagaa aaatattact gtaacagcta
gtgttgatcc tgtgattgat 1140 cttttgcaac tcgagcacca ccaccaccac cactga
1176 24 371 PRT Escherichia coli 24 Met Asn Lys Ile Leu Phe Ile Phe
Thr Leu Phe Phe Ser Ser Gly Phe 1 5 10 15 Phe Thr Phe Ala Val Ser
Ala Asp Lys Asn Pro Gly Ser Glu Asn Met 20 25 30 Thr Asn Thr Ile
Gly Pro His Asp Arg Gly Gly Ser Ser Pro Ile Tyr 35 40 45 Asn Ile
Leu Asn Ser Tyr Leu Thr Ala Tyr Asn Gly Ser His His Leu 50 55 60
Tyr Asp Arg Met Ser Phe Leu Cys Leu Ser Ser Gln Asn Thr Leu Asn 65
70 75 80 Gly Ala Cys Pro Ser Ser Asp Ala Pro Gly Thr Ala Thr Ile
Asp Gly 85 90 95 Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg
Ser Leu Ile Lys 100 105 110 Arg Glu Leu Gln Ile Lys Gly Tyr Lys Gln
Phe Leu Phe Lys Asn Ala 115 120 125 Asn Cys Pro Ser Lys Leu Ala Leu
Asn Ser Ser His Phe Gln Cys Asn 130 135 140 Arg Glu Gln Ala Ser Gly
Ala Thr Leu Ser Leu Tyr Ile Pro Ala Gly 145 150 155 160 Glu Leu Asn
Lys Leu Pro Phe Gly Gly Val Trp Asn Ala Val Leu Lys 165 170 175 Leu
Asn Val Lys Arg Arg Tyr Asp Thr Thr Tyr Gly Thr Tyr Thr Ile 180 185
190 Asn Ile Thr Val Asn Leu Thr Asp Lys Gly Asn Ile Gln Ile Trp Leu
195 200 205 Pro Gln Phe Lys Ser Asn Ala Arg Val Asp Leu Asn Leu Arg
Pro Thr 210 215 220 Gly Gly Gly Thr Tyr Ile Gly Arg Asn Ser Val Asp
Met Cys Phe Tyr 225 230 235 240 Asp Gly Tyr Ser Thr Asn Ser Ser Ser
Leu Glu Ile Arg Phe Gln Asp 245 250 255 Asp Asn Ser Lys Ser Asp Gly
Lys Phe Tyr Leu Lys Lys Ile Asn Asp 260 265 270 Asp Ser Lys Glu Leu
Val Tyr Thr Leu Ser Leu Leu Leu Ala Gly Lys 275 280 285 Asn Leu Thr
Pro Thr Asn Gly Gln Ala Leu Asn Ile Asn Thr Ala Ser 290 295 300 Leu
Glu Thr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu Ile 305 310
315 320 Ser Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala
Lys 325 330 335 Val Lys Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile
Lys Ile Thr 340 345 350 Phe Thr Pro Ser Ser Gln Thr Leu Asn Asn Lys
Gln Gln Gln Gln Gln 355 360 365 Gln Gln Trp 370 25 1185 DNA
Escherichia coli 25 atgaaaaaga tatttatttt tttgtctatc atattttctg
cggtggtcag tgccgggcga 60 tacccggaaa ctacagtagg taatctgacg
aagagttttc aagcccctcg tcaggataga 120 agcgtacaat caccaatata
taacatcttt acgaatcatg tggctggata tagtttgagt 180 cataacttat
atgacaggat tgttttttta tgtacatcct cgtcgaatcc ggttaatggt 240
gcttgcccaa cccttggaac atctggagtt caatacggta ctacaaccat aaccttgcag
300 tttacagaaa aaagaagtct gataaaaaga aatattaatc ttgcaggtaa
taagaaacca 360 atatgggaga atcagagttg cgacactagc aatctaatgg
tgttgaattc gaagtcttgg 420 tcctgtgggc attacggaaa tgctaacgga
acacttctaa atctgtatat ccctgcagga 480 gaaatcaaca aattgccttt
tggagggata tgggaggcaa ctctgatctt acgcttatca 540 agatatggcg
aagtcagtag cacccattac ggcaattata ccgtaaatat tacggttgat 600
ttaactgata aaggtaatat tcaggtatgg cttccagggt ttcacagcaa cccgcgtgta
660 gacctgaatc tgcaccctat cggtaattat aaatatagtg gtagtaattc
actcgacatg 720 tgtttctatg atggatatag tacaaacagt gatagcatgg
taataaagtt ccaggatgat 780 aatcctacct attcatctga atataatctt
tataagatag ggggcactga aaaattaccc 840 tatgctgttt cactgcttat
gggagaaaaa atattttatc cagtgaatgg tcaatcattt 900 actatcaatg
acagtagtgt actcgaaaca aactggaatc gagtaaccgc agttgctatg 960
ccggaagtta atgttccagt attatgctgg ccagcaagat tgctattaaa tgctgatgta
1020 aatgctcccg atgcaggaca gtattcagga cagatatata taacatttac
acccagtgtc 1080 gaaaatttag acaataaaca agtcgaaaaa aatattactg
tgagggcaag tgttgaccct 1140 aaacttgatc ttctgcaact cgagcaccac
caccaccacc actga 1185 26 376 PRT Escherichia coli 26 Gly Arg Tyr
Pro Glu Thr Thr Val Gly Asn Leu Thr Lys Ser Phe Gln 1 5 10 15 Ala
Pro Arg Gln Asp Arg Ser Val Gln Ser Pro Ile Tyr Asn Ile Phe 20 25
30 Thr Asn His Val Ala Gly Tyr Ser Leu Ser His Asn Leu Tyr Asp Arg
35 40 45 Ile Val Phe Leu Cys Thr Ser Ser Ser Asn Pro Val Asn Gly
Ala Cys 50 55 60 Pro Thr Leu Gly Thr Ser Gly Val Gln Tyr Gly Thr
Thr Thr Ile Thr 65 70 75 80 Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile
Lys Arg Asn Ile Asn Leu 85 90 95 Ala Gly Asn Lys Lys Pro Ile Trp
Glu Asn Gln Ser Cys Asp Thr Ser 100 105 110 Asn Leu Met Val Leu Asn
Ser Lys Ser Trp Ser Cys Gly His Tyr Gly 115 120 125 Asn Ala Asn Gly
Thr Leu Leu Asn Leu Tyr Ile Pro Ala Gly Glu Ile 130 135 140 Asn Lys
Leu Pro Phe Gly Gly Ile Trp Glu Ala Thr Leu Ile Leu Arg 145 150 155
160 Leu Ser Arg Tyr Gly Glu Val Ser Ser Thr His Tyr Gly Asn Tyr Thr
165 170 175 Val Asn Ile Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln
Val Trp 180 185 190 Leu Pro Gly Phe His Ser Asn Pro Arg Val Asp Leu
Asn Leu His Pro 195 200 205 Ile Gly Asn Tyr Lys Tyr Ser Gly Ser Asn
Ser Leu Asp Met Cys Phe 210
215 220 Tyr Asp Gly Tyr Ser Thr Asn Ser Asp Ser Met Val Ile Lys Phe
Gln 225 230 235 240 Asp Asp Asn Pro Thr Tyr Ser Ser Glu Tyr Asn Leu
Tyr Lys Ile Gly 245 250 255 Gly Thr Glu Lys Leu Pro Tyr Ala Val Ser
Leu Leu Met Gly Glu Lys 260 265 270 Ile Phe Tyr Pro Val Asn Gly Gln
Ser Phe Thr Ile Asn Asp Ser Ser 275 280 285 Val Leu Glu Thr Asn Trp
Asn Arg Val Thr Ala Val Ala Met Pro Glu 290 295 300 Val Asn Val Pro
Val Leu Cys Trp Pro Ala Arg Leu Leu Leu Asn Ala 305 310 315 320 Asp
Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile Tyr Ile 325 330
335 Thr Phe Thr Pro Ser Val Glu Asn Leu Asp Asn Lys Gln Val Glu Lys
340 345 350 Asn Ile Thr Val Arg Ala Ser Val Asp Pro Lys Leu Asp Leu
Leu Gln 355 360 365 Leu Glu His His His His His His 370 375 27 361
PRT Escherichia coli 27 Met Asn Lys Ile Leu Phe Ile Phe Thr Leu Phe
Phe Ser Ser Val Leu 1 5 10 15 Phe Thr Phe Ala Val Ser Ala Asp Lys
Ile Pro Gly Asp Glu Ser Ile 20 25 30 Thr Asn Ile Phe Gly Pro Arg
Asp Arg Asn Glu Ser Ser Pro Lys His 35 40 45 Asn Ile Leu Asn Asn
His Ile Thr Ala Tyr Ser Glu Ser His Thr Leu 50 55 60 Tyr Asp Arg
Met Thr Phe Leu Cys Leu Ser Ser His Asn Thr Leu Asn 65 70 75 80 Gly
Ala Cys Pro Thr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser Gly 85 90
95 Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys
100 105 110 Arg Glu Leu Gln Ile Lys Gly Tyr Lys Gln Leu Leu Phe Lys
Ser Val 115 120 125 Asn Cys Pro Ser Gly Leu Thr Leu Asn Ser Ala His
Phe Asn Cys Asn 130 135 140 Lys Asn Ala Ala Ser Gly Ala Ser Leu Tyr
Leu Tyr Ile Pro Ala Gly 145 150 155 160 Glu Leu Lys Asn Leu Pro Phe
Gly Gly Ile Trp Asp Ala Thr Leu Lys 165 170 175 Leu Arg Val Lys Arg
Arg Tyr Ser Glu Thr Tyr Gly Thr Tyr Thr Ile 180 185 190 Asn Ile Thr
Ile Lys Leu Thr Asp Lys Gly Asn Ile Gln Ile Trp Leu 195 200 205 Pro
Gln Phe Lys Ser Asp Ala Arg Val Asp Leu Asn Leu Arg Pro Thr 210 215
220 Gly Gly Gly Thr Tyr Ile Gly Arg Asn Ser Val Asp Met Cys Phe Tyr
225 230 235 240 Asp Gly Tyr Ser Thr Asn Ser Ser Ser Leu Glu Ile Arg
Phe Gln Asp 245 250 255 Asn Asn Pro Lys Ser Asp Gly Lys Phe Tyr Leu
Arg Lys Ile Asn Asp 260 265 270 Asp Thr Lys Glu Ile Ala Tyr Thr Leu
Ser Leu Leu Leu Ala Gly Lys 275 280 285 Ser Leu Thr Pro Thr Asn Gly
Thr Ser Leu Asn Ile Ala Asp Ala Ala 290 295 300 Ser Leu Glu Thr Asn
Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu 305 310 315 320 Ile Ser
Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala 325 330 335
Lys Val Glu Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile Asn Val 340
345 350 Thr Phe Thr Pro Ser Ser Gln Thr Leu 355 360 28 361 PRT
Escherichia coli 28 Met Asn Lys Ile Leu Phe Ile Phe Thr Leu Phe Phe
Ser Ser Val Leu 1 5 10 15 Phe Thr Phe Ala Val Ser Ala Asp Lys Ile
Pro Gly Asp Glu Asn Ile 20 25 30 Thr Asn Ile Phe Gly Pro Arg Asp
Arg Asn Glu Ser Ser Pro Lys His 35 40 45 Asn Ile Leu Asn Asp Tyr
Ile Thr Ala Tyr Ser Glu Ser His Thr Leu 50 55 60 Tyr Asp Arg Met
Ile Phe Leu Cys Leu Ser Ser Gln Asn Thr Leu Asn 65 70 75 80 Gly Ala
Cys Pro Thr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser Gly 85 90 95
Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys 100
105 110 Arg Glu Leu Gln Ile Lys Gly Tyr Lys Arg Leu Leu Phe Lys Gly
Ala 115 120 125 Asn Cys Pro Ser Tyr Leu Thr Leu Asn Ser Ala His Tyr
Thr Cys Asn 130 135 140 Arg Asn Ser Ala Ser Gly Ala Ser Leu Tyr Leu
Tyr Ile Pro Ala Gly 145 150 155 160 Glu Leu Lys Asn Leu Pro Phe Gly
Gly Ile Trp Asp Ala Thr Leu Lys 165 170 175 Leu Arg Val Lys Arg Arg
Tyr Asp Gln Thr Tyr Gly Thr Tyr Thr Ile 180 185 190 Asn Ile Thr Val
Lys Leu Thr Asp Lys Gly Asn Ile Gln Ile Trp Leu 195 200 205 Pro Gln
Phe Lys Ser Asp Ala Arg Val Asp Leu Asn Leu Arg Pro Thr 210 215 220
Gly Gly Gly Thr Tyr Ile Gly Arg Asn Ser Val Asp Met Cys Phe Tyr 225
230 235 240 Asp Gly Tyr Ser Thr Asn Ser Ser Ser Leu Glu Leu Arg Phe
Gln Asp 245 250 255 Asn Asn Pro Lys Ser Asp Gly Lys Phe Tyr Leu Arg
Lys Ile Asn Asp 260 265 270 Asp Thr Lys Glu Ile Ala Tyr Thr Leu Ser
Leu Leu Leu Ala Gly Lys 275 280 285 Ser Leu Thr Pro Thr Asn Gly Thr
Ser Leu Asn Ile Ala Asp Ala Ala 290 295 300 Ser Leu Glu Ile Asn Trp
Asn Arg Ile Thr Ala Val Thr Met Pro Glu 305 310 315 320 Ile Ser Val
Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala 325 330 335 Lys
Val Glu Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile Asn Ile 340 345
350 Thr Phe Thr Pro Ser Ser Gln Thr Leu 355 360 29 363 PRT
Escherichia coli 29 Met Lys Lys Ile Phe Ile Phe Leu Ser Ile Ile Phe
Ser Ala Val Val 1 5 10 15 Ser Ala Gly Arg Tyr Pro Glu Thr Thr Val
Gly Asn Leu Thr Lys Ser 20 25 30 Phe Gln Ala Pro Arg Leu Asp Arg
Ser Val Gln Ser Pro Ile Tyr Asn 35 40 45 Ile Phe Thr Asn His Val
Ala Gly Tyr Ser Leu Ser His Ser Leu Tyr 50 55 60 Asp Arg Ile Val
Phe Leu Cys Thr Ser Ser Ser Asn Pro Val Asn Gly 65 70 75 80 Ala Cys
Pro Thr Ile Gly Thr Ser Gly Val Gln Tyr Gly Thr Thr Thr 85 90 95
Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys Arg Asn Ile 100
105 110 Asn Leu Ala Gly Asn Lys Lys Pro Ile Trp Glu Asn Gln Ser Cys
Asp 115 120 125 Phe Ser Asn Leu Met Val Leu Asn Ser Lys Ser Trp Ser
Cys Gly Ala 130 135 140 His Gly Asn Ala Asn Gly Thr Leu Leu Asn Leu
Tyr Ile Pro Ala Gly 145 150 155 160 Glu Ile Asn Lys Leu Pro Phe Gly
Gly Ile Trp Glu Ala Thr Leu Ile 165 170 175 Leu Arg Leu Ser Arg Tyr
Gly Glu Val Ser Ser Thr His Tyr Gly Asn 180 185 190 Tyr Thr Val Asn
Ile Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln 195 200 205 Val Trp
Leu Pro Gly Phe His Ser Asn Pro Arg Val Asp Leu Asn Leu 210 215 220
Arg Pro Ile Gly Asn Tyr Lys Tyr Ser Gly Ser Asn Ser Leu Asp Met 225
230 235 240 Cys Phe Tyr Asp Gly Tyr Ser Thr Asn Ser Asp Ser Met Val
Ile Lys 245 250 255 Phe Gln Asp Asp Asn Pro Thr Asn Ser Ser Glu Tyr
Asn Leu Tyr Lys 260 265 270 Ile Gly Gly Thr Glu Lys Leu Pro Tyr Ala
Val Ser Leu Leu Met Gly 275 280 285 Glu Lys Ile Phe Tyr Pro Val Asn
Gly Gln Ser Phe Thr Ile Asn Asp 290 295 300 Ser Ser Val Leu Glu Thr
Asn Trp Asn Arg Val Thr Ala Val Ala Met 305 310 315 320 Pro Glu Val
Asn Val Pro Val Leu Cys Trp Pro Ala Arg Leu Leu Leu 325 330 335 Asn
Ala Asp Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile 340 345
350 Tyr Ile Thr Phe Thr Pro Ser Val Glu Asn Leu 355 360 30 363 PRT
Escherichia coli 30 Met Lys Lys Ile Phe Ile Phe Leu Ser Ile Ile Phe
Ser Ala Val Val 1 5 10 15 Ser Ala Gly Arg Tyr Pro Glu Thr Thr Val
Gly Asn Leu Thr Lys Ser 20 25 30 Phe Gln Ala Pro Arg Leu Asp Arg
Ser Val Gln Ser Pro Ile Tyr Asn 35 40 45 Ile Phe Thr Asn His Val
Ala Gly Tyr Ser Leu Ser His Arg Leu Tyr 50 55 60 Asp Arg Ile Val
Phe Val Cys Thr Ser Ser Ser Asn Pro Val Asn Gly 65 70 75 80 Ala Cys
Pro Thr Ile Gly Thr Ser Gly Val Glu Tyr Gly Thr Thr Thr 85 90 95
Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys Arg Asn Ile 100
105 110 Asn Leu Ala Gly Asn Lys Lys Pro Ile Trp Glu Asn Gln Ser Cys
Asp 115 120 125 Phe Ser Asn Leu Met Val Leu Asn Ser Lys Ser Trp Ser
Cys Gly Ala 130 135 140 Gln Gly Asn Ala Asn Gly Thr Leu Leu Asn Leu
Tyr Ile Pro Ala Gly 145 150 155 160 Glu Ile Asn Lys Leu Pro Phe Gly
Gly Ile Trp Glu Ala Thr Leu Ile 165 170 175 Leu Arg Leu Ser Arg Tyr
Gly Glu Val Ser Ser Thr His Tyr Gly Asn 180 185 190 Tyr Thr Val Asn
Ile Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln 195 200 205 Val Trp
Leu Pro Gly Phe His Ser Asn Pro Arg Val Asp Leu Asn Leu 210 215 220
His Pro Ile Gly Asn Tyr Lys Tyr Ser Gly Ser Asn Ser Leu Asp Met 225
230 235 240 Cys Phe Tyr Asp Gly Tyr Ser Thr Asn Ser Asp Ser Met Val
Ile Lys 245 250 255 Phe Gln Asp Asp Asn Pro Thr Asn Ser Ser Glu Tyr
Asn Leu Tyr Lys 260 265 270 Arg Gly Gly Thr Glu Lys Leu Pro Tyr Ala
Val Ser Leu Leu Met Gly 275 280 285 Gly Lys Ile Phe Tyr Pro Val Asn
Gly Gln Ser Phe Thr Ile Asn Asp 290 295 300 Ser Ser Val Leu Glu Thr
Asn Trp Asn Arg Val Thr Ala Val Ala Met 305 310 315 320 Pro Glu Val
Asn Val Pro Val Leu Cys Trp Pro Ala Arg Leu Leu Leu 325 330 335 Asn
Ala Asp Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile 340 345
350 Tyr Ile Thr Phe Thr Pro Ser Val Glu Asn Leu 355 360 31 363 PRT
Escherichia coli 31 Met Lys Lys Ile Phe Ile Phe Leu Ser Ile Ile Phe
Ser Ala Val Val 1 5 10 15 Ser Ala Gly Arg Tyr Pro Glu Thr Thr Val
Gly Asn Leu Thr Lys Ser 20 25 30 Phe Gln Ala Pro Arg Leu Asp Arg
Ser Val Gln Ser Pro Ile Tyr Asn 35 40 45 Ile Phe Thr Asn His Val
Ala Gly Tyr Ser Leu Ser His Arg Leu Tyr 50 55 60 Asp Arg Ile Val
Phe Val Cys Thr Ser Ser Ser Asn Pro Val Asn Gly 65 70 75 80 Ala Cys
Pro Thr Ile Gly Thr Ser Gly Val Glu Tyr Gly Thr Thr Thr 85 90 95
Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys Arg Asn Ile 100
105 110 Asn Leu Ala Gly Asn Lys Lys Pro Ile Trp Glu Asn Gln Ser Cys
Asp 115 120 125 Phe Ser Asn Leu Met Val Leu Asn Ser Lys Ser Trp Ser
Cys Gly Ala 130 135 140 Gln Gly Asn Ala Asn Gly Thr Leu Leu Asn Leu
Tyr Ile Pro Ala Gly 145 150 155 160 Glu Ile Asn Lys Leu Pro Phe Gly
Gly Ile Trp Glu Ala Thr Leu Ile 165 170 175 Leu Arg Leu Ser Arg Tyr
Gly Glu Val Ser Ser Thr His Tyr Gly Asn 180 185 190 Tyr Thr Val Asn
Ile Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln 195 200 205 Val Trp
Leu Pro Gly Phe His Ser Asn Pro Arg Val Asp Leu Asn Leu 210 215 220
His Pro Ile Gly Asn Tyr Lys Tyr Ser Gly Ser Asn Ser Leu Asp Met 225
230 235 240 Cys Phe Tyr Asp Gly Tyr Ser Thr Asn Ser Asp Ser Met Val
Ile Lys 245 250 255 Phe Gln Asp Asp Asn Pro Thr Asn Ser Ser Glu Tyr
Asn Leu Tyr Lys 260 265 270 Arg Gly Gly Thr Glu Lys Leu Pro Tyr Ala
Val Ser Leu Leu Met Gly 275 280 285 Gly Lys Ile Phe Tyr Pro Val Asn
Gly Gln Ser Phe Thr Ile Asn Asp 290 295 300 Ser Ser Val Leu Glu Thr
Asn Trp Asn Arg Val Thr Ala Val Ala Met 305 310 315 320 Pro Glu Val
Asn Val Pro Val Leu Cys Trp Pro Ala Arg Leu Leu Leu 325 330 335 Asn
Ala Asp Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile 340 345
350 Tyr Ile Thr Phe Thr Pro Ser Val Glu Asn Leu 355 360 32 355 PRT
Escherichia coli 32 Met Phe Leu Cys Ser Gln Val Tyr Gly Gln Ser Trp
His Thr Asn Val 1 5 10 15 Glu Ala Gly Ser Ile Asn Lys Thr Glu Ser
Ile Gly Pro Ile Asp Arg 20 25 30 Ser Ala Ala Ala Ser Tyr Pro Ala
His Tyr Ile Phe His Glu His Val 35 40 45 Ala Gly Tyr Asn Lys Asp
His Ser Leu Phe Asp Arg Met Thr Phe Leu 50 55 60 Cys Met Ser Ser
Thr Asp Ala Ser Lys Gly Ala Cys Pro Thr Gly Glu 65 70 75 80 Asn Ser
Lys Ser Ser Gln Gly Glu Thr Asn Ile Lys Leu Ile Phe Thr 85 90 95
Glu Lys Lys Ser Leu Ala Arg Lys Thr Leu Asn Leu Lys Gly Tyr Lys 100
105 110 Arg Phe Leu Tyr Glu Ser Asp Arg Cys Ile His Tyr Val Asp Lys
Met 115 120 125 Asn Leu Asn Ser His Thr Val Lys Cys Val Gly Ser Phe
Thr Arg Gly 130 135 140 Val Asp Phe Thr Leu Tyr Ile Pro Gln Gly Glu
Ile Asp Gly Leu Leu 145 150 155 160 Thr Gly Gly Ile Trp Glu Ala Thr
Leu Glu Leu Arg Val Lys Arg His 165 170 175 Tyr Asp Tyr Asn His Gly
Thr Tyr Lys Val Asn Ile Thr Val Asp Leu 180 185 190 Thr Asp Lys Gly
Asn Ile Gln Val Trp Thr Pro Lys Phe His Ser Asp 195 200 205 Pro Arg
Ile Asp Leu Asn Leu Arg Pro Glu Gly Asn Gly Lys Tyr Ser 210 215 220
Gly Ser Asn Val Leu Glu Met Cys Leu Tyr Asp Gly Tyr Ser Thr His 225
230 235 240 Ser Gln Ser Ile Glu Met Arg Phe Gln Asp Asp Ser Gln Thr
Gly Asn 245 250 255 Asn Glu Tyr Asn Leu Ile Lys Thr Gly Glu Pro Leu
Lys Lys Leu Pro 260 265 270 Tyr Lys Leu Ser Leu Leu Leu Gly Gly Arg
Glu Phe Tyr Pro Asn Asn 275 280 285 Gly Glu Ala Phe Thr Ile Asn Asp
Thr Ser Ser Leu Phe Ile Asn Trp 290 295 300 Asn Arg Ile Lys Ser Val
Ser Leu Pro Gln Ile Ser Ile Pro Val Leu 305 310 315 320 Cys Trp Pro
Ala Asn Leu Thr Phe Met Ser Glu Leu Asn Asn Pro Glu 325 330 335 Ala
Gly Glu Tyr Ser Gly Ile Leu Asn Val Thr Phe Thr Pro Ser Ser 340 345
350 Ser Ser Leu 355
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