U.S. patent application number 10/620256 was filed with the patent office on 2005-03-24 for production of recombinant lactoferrin and lactoferrin polypeptides using cdna sequences in various organisms.
Invention is credited to Conneely, Orla M., Headon, Denis R., May, Gregory S., O'Malley, Bert W..
Application Number | 20050064546 10/620256 |
Document ID | / |
Family ID | 23367308 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050064546 |
Kind Code |
A1 |
Conneely, Orla M. ; et
al. |
March 24, 2005 |
Production of recombinant lactoferrin and lactoferrin polypeptides
using cDNA sequences in various organisms
Abstract
The verified cDNA sequences for human, bovine and porcine
lactoferrin protein have been used to prepare recombinant
lactoferrin for therapeutic and nutritional applications. Regions
of the cDNA such as the Fe binding sites can be used to make an hLF
polypeptide product The present invention provides novel plasmids,
transfected eucaryotic cells and methods of producing these
plasmids and transfected eucaryotic cells. The novel plasmid
contains the cDNA for lactoferrin protein. Methods for the
production of lactoferrin protein in fungi and bacteria are also
provided. Thus, the present invention provides an efficient and
economical means for the production of recombinant lactoferrin
protein and lactoferrin related polypeptides.
Inventors: |
Conneely, Orla M.; (Houston,
TX) ; Headon, Denis R.; (Galway, IE) ;
O'Malley, Bert W.; (Houston, TX) ; May, Gregory
S.; (Houston, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
23367308 |
Appl. No.: |
10/620256 |
Filed: |
July 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10620256 |
Jul 15, 2003 |
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09633739 |
Aug 7, 2000 |
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6635447 |
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09633739 |
Aug 7, 2000 |
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08456108 |
May 30, 1995 |
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6100054 |
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08456108 |
May 30, 1995 |
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08145681 |
Oct 28, 1993 |
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5571691 |
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08145681 |
Oct 28, 1993 |
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07967947 |
Oct 27, 1992 |
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07967947 |
Oct 27, 1992 |
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07348270 |
May 5, 1989 |
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10620256 |
Jul 15, 2003 |
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07873304 |
Apr 24, 1992 |
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Current U.S.
Class: |
435/69.1 ;
435/189; 435/204; 435/254.2; 435/254.3; 435/320.1; 435/348;
536/23.2 |
Current CPC
Class: |
A61P 31/12 20180101;
A61P 43/00 20180101; A61P 31/04 20180101; C07K 14/79 20130101 |
Class at
Publication: |
435/069.1 ;
435/189; 435/204; 435/254.2; 435/254.3; 435/348; 435/320.1;
536/023.2 |
International
Class: |
C07H 021/04; C12N
009/02; C12N 009/32; C12N 001/16; C12N 001/18; C12N 005/06; C12N
015/74 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. HD27965 awarded by the National Institute of Health. The
government has certain rights in the invention.
Claims
1-23. (Canceled)
24. A process for producing lactoferrin which comprises culturing a
transformant eucaryotic cell containing a recombinant plasmid, said
plasmid comprising a plasmic vector having a
polydeoxyribonucleotide which codes for a lactoferrin proteins in a
suitable nutrient medium until the lactoferrin protein is formed
and isolating the lactoferin protein.
25-29. (Canceled)
30. A method for producing biologically active recombinant
lactoferrin comprising the steps of: combining sequences containing
a selectable marker gene, a promotor, a transcription termination
sequence, and a linker sequence; cloning said sequences to form a
plasmid; digesting said plasmid with a restriction endonuclease;
inserting a cDNA coding for human, bovine or porcine lactoferrin
into a restriction site; and transforming a cells with said plasmid
to produce said recombinant lactoferrin.
31. The method of claim 30, wherein said selectable marker gene is
selected from the group consisting of pryr4, pyrG, andS, argB and
trpC.
32. Canceled
33. The method of claim 30, wherein said promotor is selected from
the group consisting of alcohol dehydrogenase, argB,
.alpha.-amylase, glucoamylase, alcohol dehydrogenase and benA.
34. The method of claim 30, wherein said transcription termination
sequence is selected from the group consisting of .alpha.-amylase,
glucoamylase, alcohol dehydrogenase and bena.
35. The method of claim 30, wherein said linker sequence is
selected from the group consisting of .alpha.-amylase, glucoamylase
and lactoferrin.
36-57. (Canceled)
58. A method for producing biologically active recombinant
lactoferrin comprising the steps of: combining sequences containing
a selectable marker gene, a promotor, a transcription termination
sequence, and a linker sequence; cloning said sequences to form a
plasmid; digesting said plasmid with a restriction endocnuclease;
inserting a substitution analog of a cDNA sequence selected from
the group consisting of SEQ. ID No. into a restriction site; and
transforming eucaryotic cells with said plasmid expressing
lactoferrin cDNA which produces said recombinant lactoferrin.
59. The method of claim 58, wherein said selectable marker gene is
selected from the group consisting of pyr4, pyrG, andS, argB and
trpC.
60. Canceled
61. A recombinant lactoferrin produced by the method of claim
58.
62. The method of claim 58, wherein said promotor is selected from
the group consisting of alcohol dehydrogenase, argB,
.alpha.-amylase, glucoamylase, and benA.
63. The method of claim 58, wherein said linker sequence is
selected from the group consisting of .alpha.-amylase,
glucoamylase, alcohol dehydrogenase and benA.
64. The method of claim 58, wherein said linker sequence is
selected from the group consisting of .alpha.-amylase,
glucoamylase, and lactoferrin.
65-68. Canceled.
Description
RELATED APPLICATIONS
[0001] This application is a continuation in part of pending
application Ser. No. 07/967,947, filed Oct. 27, 1992, which in turn
is a continuation of application Ser. No. 07/348,270, filed May 05,
1989, now abandoned. This application is also a continuation in
part of pending application Ser. No. 07/873,304 filed Apr. 24,
1992.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
iron-binding glycoproteins. More specifically, the present
invention relates to the recombinant production of various
lactoferrins.
[0005] 2. Description of the Prior Art
[0006] Lactoferrin (LF) is an iron-binding glycoprotein found in
milk and other secretions and body fluids. It is one of a number of
iron binding proteins, sometimes referred to as transferring, and
is involved in iron binding and delivery in mammals.
[0007] Human lactoferrin (hLF) is a member of the transferrin
family of iron-binding monomeric glycoproteins. It was originally
discovered in milk where it can reach levels of 7 grams/liter in
colostrum. LF has since been detected in other external fluids of
humans and other mammals. The fluids include tears, saliva and
mucosal secretions and also in the secondary granules of
polymorphonuclear leukocytes.
[0008] Lactoferrin has been implicated as a factor in resistance
against enteritis infections in suckled newborn humans. The
bacteriocidal/bacteriostatic actions are considered to be due at
least in part to the iron binding properties of lactoferrin.
Lactoferrin decreases the iron availability to iron-requiring
microorganisms and thereby interferes with their growth and
reproduction. At least one non-ironbinding bactericidal domain has
also been reported for human lactoferrin. Lactoferrin is also
considered to have antiviral properties and to have other potential
therapeutic applications.
[0009] LF is a 78 kilo Dalion (k Da) glycoprotein having a bilobal
structure with a high degree of homology between the C and N
terminal halves which is evident at both the amino acid and three
dimensional structural level. Each of these lobes can reversibly
bind one ferric iron with high affinity and with the concomitant
binding of bicarbonate. The biological functions proposed for
lactoferrin include protection against microbial infection,
enhanced intestinal iron absorption in infants, promotion of cell
growth, regulation of myelopoiesis and modulation of inflammatory
responses.
[0010] Human lactoferrin (hLF) has a high affinity for iron and two
Fe.sup.3+ cations can be bound per molecule. The complete HLF
protein has been subjected to amino acid sequencing and is reported
to have 703 amino acids. There are two glycosylation sites.
Metz-Boutigue et al., Eur. J Biochem., 145:659-676 (1984). Anderson
et al., Proc. Nat'l Acad. Sci. USA, 84:1769-1773 (April 1987).
[0011] In other studies, a cloned cDNA probe for amino acids 428 to
703 of the Metz-Boutigue structure of the lactoferrin protein was
isolated. The cDNA sequence was in general agreement with the
earlier analysis of the amino acid sequence of the protein. Rado et
al., Blood, 79; 4:989-993, 79; 4:989-993 (October 1987). The probe
was reported to encompass approximately 40% of the coding region
and the 3' terminus. The cDNA sequence for both porcine, Lydon, J.
P., et al., Biochem. Biophysic. ACTA, 1132:97-99 (1992); Alexander,
L. J., et al., Animal Genetics, 23:251-256 (1992) and bovine
lactoferrin, Mead, P. E., et al., Nucleic Acids Research, 18:7167
(1990); Pierce, A., et al., Eur. J Biochem., 196:177-184 (1991),
have been determined.
[0012] Polypeptides derived from lactoferrin are also known to be
biologically active. A fragment containing a possible iron binding
site was reported by Rado, et al. supra. An N-terminal human
lactoferrin fragment, including a bactericidal domain of HLF, was
isolated from a pepsin digest. Bellamy, W. M., et al, Biochem.
Biophys. ACTA, 1121:130-136 (1992). Synthetic 23 and 25 amino acid
polypeptides were synthesized and found to have activities similar
to the fragments derived by pepsin digestion. The synthesis
details, yields and purity of the synthetic peptides were not
reported. Bellamy et al. do not provide a practical route to large
scale production of the polypeptides free of the contaminates
resulting form isolation from natural products.
[0013] The bactericidal domain from lactoferrin has a broad
spectrum of antimicrobial action. Bellamy, W. M. et al., J. App.
Bact. 73, 472-479 (1992). Although Bellamy et al. report that
bovine lactoferrin isolated from milk can provide commercial
quantities of the bovine polypeptide by pepsin digestion, the
materials used in both studies had a minimum purity of only 95%.
Bellamy, et al. do not provide constructs for the large scale
production of synthetic human or bovine lactoferrin or lactoferrin
polypeptides. Neither does Bellamy et al. provide the ability to
produce peptides that are not available by enzyme digestion.
[0014] Filamentous fungi have been successfully employed as hosts
in the industrial production of extracellular glycoproteins.
Certain industrial strains are capable of secreting gram quantities
of these proteins. In addition, filamentous fungi are able to
correctly perform post-translational modifications of eucaryotic
proteins and many strains have U.S. Food and Drug Administration
approval. Furthermore, large scale fermentation technology and
downstream processing experience is available.
[0015] Currently, there is no efficient and economical way to
produce hLF, other species lactoferrin, or to control production of
lactoferrin polypeptides. Consequently, a long felt need and
description in this art would be met by the development of an
efficient method for the production of human lactoferrin for
nutritional and therapeutic applications and for further
investigation into its mechanism of action.
SUMMARY OF THE INVENTION
[0016] The invention comprises the verified cDNA sequences for
human lactoferrin, and cDNA expression systems for use of various
lactoferrin DNA sequences to produce human, bovine, porcine and
other lactoferrins for a variety of end uses. The cDNA expression
systems of the invention also provide a practical route and method
to make lactoferrin polypeptides or fragments having biological
activity. The hLF cDNA includes an open reading frame of 2133
nucleotides coding for a protein of 711 amino acids. These 711
amino acids include 19 amino acids corresponding to a secretion
signal peptide sequence followed by 692 amino acids of mature human
lactoferrin. The cDNA sequence and deduced amino acid sequence
differ from the previously published data of Metz-Boutigue,
supra.
[0017] In one embodiment, the present invention provides for a
recombinant plasmid comprising the cDNA of human or other
lactoferrin. The plasmid of the present invention is adapted for
expression in a eucaryotic cell and contains the regulatory
elements necessary for the expression of the human lactoferrin cDNA
in this eucaryotic cell.
[0018] In another embodiment, the present invention provides for a
transformed cell which includes a heterologous DNA sequence which
codes for lactoferrin or a polypeptide related to lactoferrin. The
heterologous DNA sequence will preferably be incorporated into a
plasmid. Eucaryotic host cells are selected from the group
consisting of mammalian cells, immortalized mammalian cells, fungi
or yeasts. Preferred cells include filamentous fungi comprising
Aspergillus, and yeasts. The plasmid contains a plasmid vector into
which a polydeoxyribonucleotide (DNA) segment coding for human or
other lactoferrin protein has been inserted.
[0019] In yet another embodiment of the present invention, there is
provided a process for producing recombinant human or other
lactoferrin which comprises culturing a transformant eucaryotic
cell, which includes a recombinant plasmid. The plasmid contains a
plasmid vector having a polydeoxyribonucleotide coding for the
lactoferrin protein. After culturing in a suitable nutrient medium
until lactoferrin protein is formed, the lactoferrin protein is
isolated.
[0020] In still yet another embodiment of the present invention,
there is provided a recombinant expression vector. This vector
comprises a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression; (2) cDNA coding for lactoferrin; (3) appropriate
transcription and translation initiation and termination sequences;
and (4) a genetic element for selection of transformed cells or
spores such as Aspergillus spores that have been transformed with
the vector.
[0021] In still yet another embodiment of the present invention,
there is provided a method for producing biologically active
recombinant lactoferrin. The method comprises synthesizing
sequences containing a selectable marker gene, a promotor, a
transcription termination sequence, and a linker sequence; cloning
the sequences to form a plasmid; digesting the plasmid with a
restriction endonuclease; inserting a cDNA coding for lactoferrin
into a restriction site; and transforming eucaryotic cells with the
plasmid expressing lactoferrin cDNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] So that the manner in which the above recited features,
advantages, and objects of the invention, as well as others which
will become clear, are obtained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a
part of this specification.
[0023] It is to be noted, however, that the appended drawings
illustrate preferred embodiments of the invention and therefore not
to be considered limiting of its scope. The invention may admit to
other equally effective equivalent embodiments.
[0024] FIG. 1 is a schematic drawing of the hLF cDNA including the
locations of the 5' untranslated region, the secretion peptide
signal sequence, mature lactoferrin and 3' untranslated region.
[0025] FIG. 2 is the cDNA sequence (SEQ. ID No. 1) with deduced
amino acids (SEQ. ID No. 2) for the human lactoferrin protein and
signal peptide sequence.
[0026] FIG. 3 is a schematic representation of an autoradiograph of
recombinant human lactoferrin protein expressed from the complete
cDNA.
[0027] FIG. 4 is a schematic representation of an autoradiograph of
the results of in vitro translation of a 2,140 bp human lactoferrin
sequence and hLF protein in reticulocyte lysates.
[0028] FIG. 5 depicts a schematic representation of the Aspergillus
oryzae expression plasmid, pAhlfg.
[0029] FIG. 6 shows a southern blot analysis of transformed
Aspergillus oryzae strains.
[0030] FIG. 7 depicts an RNA analysis of transformant versus
control A07.
[0031] FIG. 8 shows the silver stained SDS-acrylimide gel analysis
of recombinant LF secretion and purification.
[0032] FIG. 9 illustrates the characterization of recombinant human
LF.
[0033] FIG. 10 is a western immunoblot of cellular extracts of
transformed E.coli cells expressing the C terminal fragment of
LF.
[0034] FIG. 11 shows the coomassie-stained SDS-PAGE analysis of
extracts of transformed E. coli cells expressing the C terminal
fragment of LF.
[0035] FIG. 12 shows the expression and purification of the
glutathione S-transferase/LFN-1 fusion protein.
[0036] FIG. 13 Schematic representation of the A. Oryzae universal
expression plasmid, pAG.
[0037] FIG. 14 is the (A) cDNA sequence (SEQ. ID No. 3) with (B)
deduced amino acids (SEQ. ID No. 4) for the bovine lactoferrin
protein.
[0038] FIG. 15 is the (A) cDNA sequence (SEQ. ID No. 5) with (B)
deduced amino acids (SEQ. ID No. 6) for the porcine lactoferrin
protein.
[0039] FIG. 16 is a Western blot showing hLF expression in
Saccharomyces Cervisiae.
[0040] FIG. 17 is a schematic of the plasmid used for expression of
the cDNA (SEQ. ID No. 1) in Aspergillis Nidulans.
[0041] FIG. 18 shows restriction enzyme cleavage sites for the
human cDNA sequence.
[0042] FIG. 19 shows restriction enzyme cleavage sites for the
bovine cDNA sequence.
[0043] FIG. 20 shows restriction enzyme cleavage sites for the
porcine cDNA sequence.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0044] For the purposes of the present application, the term
"transferrin family" means a family of iron transferring proteins
including serum transferrin, ovotransferrin and lactoferrin. These
proteins are all structurally related.
[0045] For the purposes of the present application, the term
"vector(s)" means plasmid, cosmid, phage or other vehicle to allow
insertion, propagation and expression of lactoferrin cDNA.
[0046] For the purposes of the present application, the term
"host(s)" means any cell that will allow lactoferrin
expression.
[0047] For the purposes of the present application, the term
"promotor(s)" means regulatory DNA sequences that controls
transcription of the lactoferrin cDNA.
[0048] For the purposes of the present application, the term
"multiple cloning cassette" means a DNA fragment containing
restriction enzyme cleavage sites for a variety of enzymes allowing
insertion of a variety of cDNAs.
[0049] For the purposes of the present application, the term
"transformation" means incorporation permitting expression of
heterologous DNA sequences by a cell.
[0050] For the purposes of the present application, the term "iron
binding capacity" means ability to bind Fe. Fully functional human
lactoferrin can bind two atoms of iron per molecule of LF.
[0051] For the purposes of the present application, the term
"biological activity/biological active" means biological activity
of lactoferrin as measured by its ability to bind iron, or kill
microorganisms, or retard the growth of microorganisms, or to
function as an iron transfer protein.
[0052] For the purposes of the present application, the term
"substitution analog" referring to a DNA sequence means a DNA
sequence in which one or more codons specifying one or more amino
acids of lactoferrin or a lactoferrin polypeptide are replaced by
alternate codons that specify the same amino acid sequence with a
different DNA sequence. Where "substitution analog" refers to a
protein or polypeptide it means the substitution of a small number,
generally five or less, commonly 3 or 4, and more often 1 or 2
amino acids as are known to occur in allelic variation in human and
other mammalian proteins wherein the biological activity of the
protein is maintained. For example, hLF isolated from milk has been
reported to differ from the hLF of SEQ. ID No. 2 at two amino acid
residues.
[0053] The confirmation of the cDNA sequence and the deduced amino
acid have been proven by multiple confirmation procedures. These
are:
[0054] 1. Multiple sequence analyses.
[0055] 2. Comparison of the amino acid sequence deduced from the
cDNA with that of hLF generated by conventional amino acid
sequencing of hLF isolated from milk. The unique cDNA sequence
which encodes the human lactoferrin protein has a variety of
applications as known and indicated in the literature.
[0056] 3. Transcription and translation of hLF protein from the
cDNA with positive identification using an anti-hLF antibody.
[0057] The cDNA sequence of the present invention can be used to
prepare recombinant human lactoferrin, thus making available a
source of protein for therapeutic and nutritional applications. The
confirmed cDNA of this invention can be used in an appropriate
cloning vehicle to replicate the cDNA sequence. Also, the cDNA can
be incorporated into a vector system for human lactoferrin
expression. Other lactoferrin DNA sequences can be substituted for
the human lactoferrin cDNA sequence to provide bovine, porcine,
equine or other lactoferrins. Partial cDNA sequences can also be
employed to give desired lactoferrin derived polypeptides. The
expression systems of the invention can be used to provide
lactoferrin derived polypeptides that are not available by
enzymatic digestion of naturally occurring lactoferrin. The
invention further provides an expression system for producing
lactoferrin and lactoferrin related polypeptides in mammalian cell
lines, other eucaryotic cells including yeast and fungal cells and
procaryotic cells. The invention allows for the production of
lactoferrin free of lactoperoxidase, lysozyme, or other proteins
that are contaminants of lactoferrin isolated from milk or other
natural products. This invention is not limited to any particular
uses of the human cDNA sequence or production of lactoferrin of
other species from the appropriate DNA sequences.
[0058] The recombinant LF being a protein derived by recombinant
techniques can be used in a variety of applications. The human gene
can be transferred to mammalian systems such as cows and other
agriculturally important animals and expressed in milk. The
incorporation of a human lactoferrin gene and expression in the
milk of animals can combat an iron deficiency typical in piglets.
The inclusion of the human lactoferrin gene with expression should
improve an animal's disease resistance to bacterial and viral
infection. The tissue specific expression of human lactoferrin in
mammary glands, for instance, would impart the bacteriocidal and
virucidal benefit of the expressed gene to young feeding on the
milk and would provide a production means for the secreted protein
for therapeutic use.
[0059] The gene can be placed in the appropriate cloning vector for
the production of LF. The LF produced by recombinant methods can be
used in a variety of products including human or animal foods, as
therapeutic additives to enhance iron transport and delivery, and
for the virucidal and bacteriocidal qualities, as additives for
eyedrops, contact lens and other eye care solutions, topical skin
care products, eardrops, mouthwashes, chewing gum and toothpaste.
The recombinant LF would provide a safe, naturally occurring
product which can be topically applied as well as ingested safely.
The bactericidal lactoferrin polypeptides are useful as
preservatives in the above listed products, and as therapeutic
anti-infection agents. The iron binding polypeptides are useful as
iron carrier proteins for nutritional and therapeutic uses, and as
bacteriostats and bactericides, especially in products of the types
listed above. Each protein may also be used as a nutrition
supplement and as a source of amino acids.
[0060] The full-length cDNA encoding human lactoferrin has been
isolated, and the analysis has been completed. The cDNA sequence
has been confirmed as human lactoferrin cDNA by comparison of the
deduced amino acid sequence with the published amino acid sequence
of hLF. The expression of lactoferrin was observed in a eucaryotic
expression system from the cDNA and a plasmid vector. The presence
of lactoferrin was confirmed by standard Western immunoblot
analysis using anti-human lactoferrin antibodies and relative
molecular mass measurement.
[0061] FIG. 1 is a schematic of the lactoferrin cDNA. The sequence
can generally be described as an initial 5' untranslated region, 17
nucleotides in length. The next portion is 57 nucleotides which
codes for the 19 amino acid secretion signal peptide starting with
methionine. The next sequence of the cDNA codes for the mature
human lactoferrin protein of 692 amino acids followed by the 3'
untranslated region of 208 nucleotides which ends the cDNA. The
complete sequence is 2,358 nucleotides in length. The hLF protein
contains glycosylation sites. The hLF protein with secretion signal
sequence has an expected molecular mass of 78,403 daltons and the
mature hLF is 76,386 daltons without added carbohydrate from
glycosylation.
[0062] FIG. 2 is the cDNA sequence (SEQ ID No. 1) with the deduced
amino acids (SEQ ID No. 2) for the secretion signal peptide and the
mature human lactoferrin protein. The numbers on FIG. 2 correspond
to the nucleotides starting at the 5' end. There are binding sites
for two iron atoms with four amino acids participating in the
binding of each iron. The amino acids at positions Asp80, Tyr112,
Tyr209, and His273 are required for coordination with one iron, and
amino acids at positions Asp415, Tyr455, Tyr548, and His6l7 bind
the other. There are two glycosylation sites at positions Asn157
and Asn498. The numbers refer to the deduced amino acid sequence.
There are 25 amino acids per line of protein sequence (starting at
nucleotide 18).
[0063] The nucleotide sequence analysis was performed on cDNA
isolated from a human prostate cDNA library. The prostate cDNA
library yielded a 2,140 bp cDNA which contained the complete 5' end
including the untranslated portion and the signal sequence. The 3'
end including the three amino acids at the carboxy terminal and the
untranslated region were obtained as a 208 bp cDNA from both a
monocyte cDNA library and human prostate cDNA library.
[0064] The data in FIG. 2 displays the full-length cDNA sequence of
this invention. The complete sequence including the 5' untranslated
region and signal peptide have not been reported. Further, the
previously reported amino acid sequence varies from the deduced
amino acid sequence for hLF of this invention. The following TABLE
1 is a summary of the differences of the amino acid sequence of the
present invention and those reported by Metz-Boutigue et al., Eur.
J. Biochem., vol. 145, pp. 659-76 (1984). For the purpose of this
table, the numbering of the amino acids will be initiated with
methionine at the start of the signal peptide sequence as amino
acid #1.
1TABLE 1 COMPARISON OF AMINO ACID SEQUENCES HUMAN LACTOFERRIN Amino
Acid Deduced Metz-Boutigue from cDNA of hLF Change Sequence # 30
Thr Substitution Ala # 48 Arg Substitution Lys # 141 Arg Insertion
NONE # 170 Ala Insertion NONE # 204 Ser Substitution Leu # 206 Gln
Substitution Lys # 209 Tyr Substitution Lys # 386 Glu Substitution
Gln # 392 Ser Substitution Trp # 410 Asp Substitution Asn # 411-424
Deletion 13 Amino acids in protein sequence not in deduced amino
acid sequence from cDNA # 532 Gln Substitution Glu # 695 Lys
Substitution Arg
[0065] FIG. 3 is the expression of human lactoferrin protein from
the complete hLF cDNA. In addition to using the entire cDNA
sequence and deduced amino acid sequence, a polypeptide of less
than the entire protein can be of value. For instance, the region
between amino acids 74-275 contains an iron binding domain which
may be used without the rest of the protein for biologically
available iron or the bacteriostatic qualities.
[0066] The cDNA sequence has been confirmed to encode lactoferrin.
The hLF cDNA was shown to encode lactoferrin by expression of the
cDNA in a eucaryotic expression system and detection of the
expressed lactoferrin protein by Western immunoblot analysis using
specific lactoferrin antibodies.
[0067] Recombinant production of lactoferrin protein has been
described below in its preferred embodiments. However, it is also
produced in number of other sources such as fungal sources such as
Saccharomyces cerevisiae, Kluyveromyces lactis, or Pichia
pastorsis, or insect cells such as SF9, or bacterial cells such as
Escherichia coli, or Bacillus, subtilis.
[0068] In one embodiment of the present invention, biologically
active recombinant lactoferrin protein is produced. This method
comprises synthesizing sequences containing a selectable marker
gene, a promotor, a transcription termination sequence and a linker
sequence.
[0069] Subsequently, the sequences are cloned to form a plasmid and
the plasmid is digested with a restriction endonuclease. A cDNA
coding for lactoferrin is inserted into a restriction site and
eucaryotic cells are then transformed with the plasmid expressing
the lactoferrin cDNA.
[0070] The selectable marker gene useful in the method of the
present invention may be any that permits isolation of cells
transformed with a lactoferrin cDNA plasmid. Preferably, the
selectable marker gene is selected from pyr4, pyrG, argB, trpC and
andS.
[0071] The promotor useful in the present invention may be any that
allows regulation of the transcription of the lactoferrin cDNA.
Preferably, the promotor is selected from the group of alcohol
dehydrogenase, argB, .alpha.-amylase and glucoamylase genes.
[0072] The transcription termination sequence useful in the present
method may be any that allows stabilization of the lactoferrin
mRNA. Preferably, the transcription termination sequence is derived
from the .alpha.-amylase, glucoamylase, alcohol dehydrogenase or
benA genes.
[0073] The linker sequence useful in the present method may be any
that contains a translation initiation codon, a secretory signal
and a restriction enzyme cleavage site. Preferably, the linker
element is derived from the .alpha.-amylase or glucoamylase
genes.
[0074] The cells, preferably eucaryotic cells, useful in the
present invention are any that allow for integration of a vector,
preferably a plasmid comprising the lactoferrin cDNA and expression
of the lactoferrin cDNA. Preferably, the eucaryotic cells are
fungal cells or insect cells. Insect cells such as SF9 are useful
in the method of the present invention. More preferably, the fungal
cells are yeast cells or Aspergillus. Most preferably, the
eucaryotic cells useful in the present invention are Aspergillus
strains, such as A. oryzae, A. niger, A. nidulans and A.
awamori.
[0075] The invention also comprises partial sequences of the cDNA
of SEQ ID No. 1, 3 and 5 and substitution analogs thereof which
code for biologically active polypeptides having homology with a
portion of lactoferrin, especially those that are not available
from enzyme digests of natural lactoferrins, the method of making
polypeptides by use and expression of partial cDNA sequences, and
the polypeptide products produced by the methods of this invention.
The desired partial sequences can be produced by restriction enzyme
cleavage, as for example at the cleavage sites indicated in FIGS.
18, 19 and 20. the partial sequences may also be synthesized or
obtained by a combination of cleavage, ligation and synthesis, or
by other methods known to those skilled in the art.
[0076] Recombinant production of lactoferrin protein and
polypeptides has been described in its preferred embodiment.
However, it is also, produced in a number of other sources such as
fungal sources such as Saccharomyces cerevisiae, Kluyveromyces
lactis, or Pichia pastorsis or insect cells such as SF9, and
lactoferrin polypeptides may also be produced in bacterial cells
such as Escherichia coli, or Bacillus subtilis.
[0077] The following examples are given for the purposes of
illustrating various embodiments of the present invention and are
not meant to be limitations of the present invention in any
form.
EXAMPLE 1
Human Lactoferrin cDNA
[0078] The complete 2,358 bp hLF cDNA was ligated to the eucaryotic
expression vector, p91023(B) at the EcoRI site downstream from the
adenovirus major late promoter. This plasmid vector was provided by
Genetics Institute (Cambridge, Mass.) and has been described in
previous publications (Wong et al., Science 288:810-815 (1985)).
The hLF cDNA expression vector was transferred into COSM-6 monkey
kidney cells using standard tissue culture transfection conditions
(Wigler et al., Cell, 16:777-785 (1979)). These COS cells do not
normally express lactoferrin. Forty-eight hours after transfection,
the cells were harvested and crude cell extracts were prepared.
Positive identification of the human lactoferrin was made by
standard Western immunoblot analysis of the proteins expressed in
the cell extracts, as well as those secreted into the cell growth
medium using a commercially available antibody directed against
human lactoferrin (Sigma). Proteins which bound to the
anti-lactoferrin antibody were detected using radio-iodine labelled
Protein A which reacts with the antibody. The immunoblots were
autoradiographed to identify the human lactoferrin protein. FIG. 3
is an autoradiographic film showing the human lactoferrin expressed
in four cell extracts prepared from tissue culture cells which were
transfected with the lactoferrin cDNA expression vector (lanes 5 to
8). Lanes 5 to 8 show that the transfected cells all contain human
lactoferrin (marked with an arrow) which is immunoreactive with the
anti-lactoferrin antibody and is the same molecular weight as human
lactoferrin (M.sub.r=78,403 daltons). The control cells which were
not transfected with the cDNA did not contain lactoferrin (lanes 3
and 4). Analysis of the growth medium showed that human lactoferrin
was also secreted into the medium from transfected cells (lane 2)
but not from control cells (lane 1).
[0079] The cDNA encodes a recombinant human lactoferrin protein
which is similar to human lactoferrin protein isolated from milk as
determined by molecular size comparisons and immunoreactivity with
anti-human lactoferrin. Furthermore, the secretion signal peptide
sequence is functional since the human lactoferrin is secreted into
the growth medium of tissue culture cells which express the
cDNA.
[0080] FIG. 4 is a schematic representation of the human
lactoferrin protein precipitated after in vitro transcription and
translation of the human lactoferrin cDNA. The 2140 bp cDNA was
from the human prostate cDNA library and included the 5'
untranslated region and the rest of the base pairs correlative to
the cDNA sequence of FIG. 2 omitting the last 208 bp at the 3'
terminus. The 2140 bp cDNA was ligated to the EcoRI site of the
plasmid vector pGEM.sub.4 (commercially available from Promega
Biotech., Madison, Wis. 53711-5305) downstream from the SP.sub.6
promoter. The plasmid construct was linearized at the 3' end of the
hLF cDNA using the restriction enzyme Hinc II or Xba I. The linear
DNA template was then transcribed in vitro using purified SP.sub.6
RNA polymerase in the presence of ribonucleotides as described in
the manufacturers protocol (Promega Corporation 1988/1989 Catalogue
and Applications Guide). The resultant mRNA was translated using
100 ng mRNA template and micrococcal nuclease treated rabbit
reticulocyte lysate (as described by Promega) in the presence of 75
uCi .sup.35S methionine (800 ci/mmol, Amersham). In vitro
synthesized lactoferrin was immunoprecipitated by incubating 100 ul
aliquots of translation reaction with 10 ug of rabbit anti-human
lactoferrin IgG (Sigma Chemical Company, St. Louis, Mo. 63178) for
2 hours at 4.degree. C. in 50 mM Tris, pH7.5/0.15M NaCl/0.05%
Tween-20 (1P buffer). The reaction volume was 200 ul.
Immunoreactive lactoferrin was precipitated after incubation for 1
hour with 50 ug of Protein A sepharose (Pharmacia, Upsalla,
Sweden). Immunoprecipitation was carried out by centrifugation for
5 minutes at 10,000 g and the precipitate was washed 5 times with 4
volumes of 1P buffer. Total translation products and
immunoprecipitates were then subjected to electrophoresis in
denaturing 7.5% polyacrylamide gels. After fixing in 50% methanol,
the gels were incubated in En.sup.3Hance (NEN, DuPont, Wilmington,
Del. 19801) for 1 hour and washed with distilled H.sub.2O. The gel
was then dried under vacuum and exposed to Kodak X-OMAT XAR film at
-70.degree. C.
[0081] Lane 1 shows .sup.14C protein molecular weight markers used
to estimate the size of the translated proteins. Lane 2 is a
negative control which shows that no .sup.35S labelled proteins are
translated in this system when no mRNA is added to the translation
mix. Lanes 3 and 4 show the total translation products obtained
when lactoferrin MRNA is added after preparation from two separate
DNA templates. The major protein band (marked with an arrow) is
human lactoferrin. This is the only band detected when the
translation products are immunoprecipitated with anti-human
lactoferrin before applying the protein to the gel (lane 6). The
measurement of molecular mass by SDS-PAGE does not correspond to
exact molecular weight due to secondary protein structure. However,
the values are shifted in a correlative manner in comparison to the
control. Analysis of the size of the translated lactoferrin is
shown in FIG. 4. The protein migrated at the expected molecular
mass of human lactoferrin (about 78 Kd). The major bands in lanes 3
and 4 which migrate higher than the 68 Kd marker band in the
control lane correspond to expected molecular mass of hLF protein
on SDS-PAGE.
EXAMPLE 2
Fungal Strains and Transformation
[0082] The pyrG mutant strain used in these studies was derived
from A. oryzae (A07 11488). The pyrG gene from A. oryzae was
mutated with 4-nitroquinoline-1-oxide. The Aspergillus
transformation was carried out by, a modification of the procedure
of Osmani, et al., J. Cell. Biol. 104:1495-1504 (1987). Conidia
(1X10.sup.6/ml) were inoculated into 50 ml of YG medium (0.5% yeast
extract 2% glucose) containing 5 mM uracil and 10 mM uridine.
Growth was at 32.degree. C. for 14-16 hours until a germ tube was
visible. The germinated conidia were harvested by centrifugation
and resuspended in 40 ml of lytic mix containing 0.4 M ammonium
sulphate, 50 mM potassium citrate (pH 6.0), 0.5% yeast extract,
0.12 g novozyme, 0.1 g Driselase, 100 .mu.l .beta.-glucuronidase,
0.5% sucrose and 10 mM MgSO.sub.4. Protoplasting was for 2-3 hours
at 32.degree. C. and 150 rpm. Following protoplasting, filtration
using sterile miracloth was necessary to remove any undigested
mycelia. The protoplasts were harvested by centrifugation and
washed twice with 10 ml of 0.4 M ammonium sulphate, 1% sucrose and
50 mM potassium citrate (pH 6.0) at 4.degree. C., resuspended in 1
ml of 0.6 M KCl; 50 mM CaCl; 10 mM Tris-HCl (pH 7.5) and placed on
ice. The transformation was performed immediately following the
protoplast preparation. Aliquots (100 .mu.l) of the protoplast were
added to 3 .mu.g of DNA and 50 .mu.l of 40% polyethylene glycol
(PEG) 6000, 50 mM CaCl.sub.2, 0.6 M KCl and 10 mM Tris-HCl, (pH
7.5). The samples were incubated on ice for fifteen minutes after
which an additional 1 ml of the PEG solution was added and
incubation at room temperature was continued for thirty minutes.
Aliquots of this mixture were plated in 3 mls of 0.7% minimal
media, supplemented with 0.4% ammonium sulphate onto plates
containing the same but solidified with 2% agar. All subsequent
growth was at 32.degree. C.
EXAMPLE 3
Plasmid Construction
[0083] A schematic representation of the expression plasmid is
shown in FIG. 5. The complete cDNA encoding human LF was repaired
using the Klenow fragment of DNA polymerase I and subcloned into
Acc I digested and repaired pGEM4 to generate pGEMhLFc. In order to
remove the LF signal sequence and generate a 5' end in frame with
the a-amylase sequences, a 252 base pair lactoferrin fragment (nt
69-321) containing Hind II/Acc I ends was obtained by polymerase
chain reaction (PCR) amplification of pGEMhLFc plasmid DNA. The
oligo primers used were as follows: the 5' end oligonucleotide as
shown in SEQ. ID. No. 7:
2 (CTGGGTCGACGTAGGAGAAGGAGTGTTCAGTGGTGC)
[0084] and the 3' end oligonucleotide as shown in SEQ. ID. No.
8:
3 (GCCGTAGACTTCCGCCGCTACAGG).
[0085] This PCR fragment was digested with Hind II and Acc I and
was subcloned into Hind II/Acc I digested pGEMhLFc generating
pGEMhLF. A 681 base pair .alpha.-amylase fragment with Asp718/Pvu
II ends encoding the promotor, signal sequence and the alanine
residue from the start of the mature .alpha.-amylase II gene, was
obtained by PCR amplification of A. oryzae genomic DNA. The oligo
primers were as follows: the 5' end oligonucleotide as shown in
SEQ. ID. No. 9:
4 (GAGGTACCGAATTCATGGTGTTTTGATCATTTTAAATTTTTATAT)
[0086] and the 3' end oligonucleotide as shown in SEQ. ID. No.
10:
5 (AGCAGCTGCAGCCAAAGCAGGTGCCGCGACCTGAAGGCCGTAC).
[0087] The amplified DNA was digested with Asp718 and Pvu II and
subcloned into Asp718/Hind II digested pGEMhLF. The resulting
plasmid (pGEMAhLF) was digested with EcoR I and the resulting 2.8
kb .alpha.-amylase-lactofe- rrin fragment was subcloned into a
unique EcoR I site in pAL3 according to the method of generating
pAhLF*. Synthetic oligonucleotides were used to provide the last
five carboxy terminal codons of lactoferrin (nt 2138-2153) missing
in pAhLF* and also to provide the first 180 bp of 3' untranslated
sequences from the A. niger glucoamylase gene. The resulting
plasmid (pAhLFG) was used to transform the A. oryzae pyrG mutant
strain.
[0088] With reference to FIG. 5, Aspergillus oryzae expression
plasmid, pAhLFG contains 681 bp of 5'-flanking sequence of the A.
oryzae AMY II gene which includes the signal sequence and first
codon of mature .alpha.-amylase. The cDNA coding for mature human
lactoferrin is subcloned in frame downstream from these sequences
allowing recombinant protein production by the addition of starch
to the growth medium. The Aspergillus niger glucoamylase 3'
untranslated region provides the transcription terminator and
polyadenylation signals. The plasmid also contains the Neurospora
crassa pyr4 selectable marker and an ampicillin resistance
gene.
[0089] The plasmid construct (pAhLFG) used for expression of human
LF contains a 681 bp fragment that encodes the promotor and
secretory signal peptide of the A. oryzae .alpha.-amylase II gene
(AMY II). The signal sequence also contains the codon for alanine
from the start of the ct-amylase mature protein generating the
signal sequence cleavage site (Leu Ala) recognizable by an
endogenase .alpha.-amylase peptidase. A human lactoferrin cDNA
fragment encoding the mature protein was subcloned in frame
immediately downstream from the AMY II sequences, placing it under
the control of this highly efficient starch inducible promoter. In
order to stabilize the transcribed human LF mRNA, a 180 bp fragment
encoding the 3' untranslated region of the glucoamylase gene from
Aspergillus niger was ligated into a unique BamH I site in the
multiple cloning cassette, immediately downstream of the human LF
cDNA providing the transcription terminator and polyadenylation
signals. The plasmid also contains the Neurospora crassa pyr4
selectable marker which complements a pyrg auxotrophic mutation of
A. oryzae and allows for selection of spores that have been
transformed with the plasmid by growth in the absence of
uridine.
EXAMPLE 4
Genomic DNA Manipulation
[0090] A. oryzae DNA was isolated from 200 mg of lyophilized
mycelia as described by Rasmussen, et al., J. Biol. Chem.,
265:13767-13775 (1990). The DNA was digested with EcoR I, size
fractionated on a 0.8% agarose gel and transferred to
nitrocellulose. Prehybridization and hybridization of the
nitrocellulose filter for Southern analysis were performed in
6.times.SSC, 0.1% SDS and 0.5% dried milk at 65.degree. C. for 16
hours. Hybridization solution contained 1.times.10.sup.7 cpm
.sup.32P-labelled lactoferrin cDNA probe (2.1 Kb). The filter was
washed in 2.times.SSC, 0.5% SDS at room temperature for 30 minutes
followed by two washes in 0.5.times.SSC, 0.5% SDS at 68.degree. C.
for 30 minutes. The filter was dried, exposed at -70.degree. C. for
two hours and developed by autoradiography.
[0091] With reference to FIG. 6, Southern blot analysis was
performed on transformed Aspergillus oryzae strains. Genomic DNA
from individual transformants and control AO7 were hybridized with
a radiolabelled hLF cDNA probe (2.1 kb). The arrow points to a
radiolabelled fragment (2.8 kb) generated upon EcoR I digestion of
the expression plasmid which is present in all the transformants
(#1-9) but is absent in control untransformed AO7. Molecular
weights of bacteriophage lambda Hind III fragments are indicated at
the left.
EXAMPLE 6
Northern Analysis
[0092] RNA was isolated from lyophilized mycelia (200 mg) using
commercially available RNazol B (Biotecx Laboratories, INC,
Houston, Tex.) according to the manufacturers instructions. Total
RNA (20 .mu.g) was electrophoresed in a 0.8% agarose gel containing
2.2 M formaldehyde. The RNA was transferred to nitrocellulose and
hybridized with either a 2.1 kb lactoferrin cDNA or a 1.8 kb
genomic .alpha.-amylase fragment corresponding to the coding region
of the .alpha.-amylase II gene. The probes were .sup.32P-labelled
by nick translation (specific activity 2.times.10.sup.8 cpm/ug).
Hybridization was carried out 2.times.SSC, 0.05% dried milk at
65.degree. C. over an ice with 2.times.10.sup.6 cpm probe/ml.
[0093] Washes were identical to those employed in the Southern
analysis. The filters were dried, exposed at -70.degree. C. for two
hours and developed by autoradiography. RNA dot blots were
performed using nitrocellulose membrane and the manifold dot blot
system. Hybridization and washing conditions were as described
above for Southern analysis. Radioactivity was quantitated using
the betagon blot analyzer.
[0094] With reference to FIG. 7, RNA analysis of transformant
versus control AO7 was performed. In Panel A, Northern analysis of
RNA (20 .mu.g) from control AO7 and transformant #1 were hybridized
with radiolabelled human LF cDNA. Human LF mRNA (2.3 kb) was
detected in the transformant #1 but not in the control
untransformed AO7. The positions of the 28S and 18S rRNA bands are
indicated on the left. In Panel B, Dot blots of RNA (5 and 10
.mu.g) from control AO7 versus transformant #1 using a
radiolabelled .alpha.-amylase genomic DNA probe. In Panel C, Dot
blots of RNA (5 and 10 .mu.g) from control A07 and transformant #1
using radiolabelled human LF cDNA probe as illustrated.
[0095] Northern analysis was performed to determine if lactoferrin
mRNA was transcribed correctly and efficiently in A. oryzae under
the regulatory control elements of the expression plasmid. Spores
(1.times.10.sup.6/ml) from transformant #1 and from control
untransformed spores were inoculated into fungal medium containing
1.5% glucose. as carbon source and grown at 30.degree. C. for 48
hours in small shake flask cultures. The cultures were washed and
reinoculated into fungal medium containing 3% starch to induce
transcription of the human LF MnRNA. After 24 hours, the cells were
harvested and RNA was isolated. Total RNA (20 .mu.g) was size
fractionated on a 1.0% agarose gel containing 2.2 M formaldehyde
and blotted on nitrocellulose.
[0096] Human lactoferrin mRNA was detected using .sup.32p labelled
human LF cDNA (2.0 kb) probe. Hybridization with human LF
radiolabelled cDNA probe detected a specific radiolabelled band at
the correct size for lactoferrin MRNA (2.3 kb) in the transformant
but not in the control untransformed strain (FIG. 7A). Quantitation
of mRNA levels by dot assay showed comparable levels of expression
of endogenous .alpha.-amylase rRNA between control AO7 and
transformant #1 (FIG. 7B). In addition, similar levels of
expression of .alpha.-amylase and human LF mRNA were seen in
transformant #1 (FIG. 7B and 7C).
EXAMPLE 6
Purification of Recombinant Human LF
[0097] LF was purified from the growth medium using CM Sephadex C50
essentially as described by Stowell, et al., Biochem J., 276:349-59
(1991). The column was pre-equilibrated with 500 ml of 0.025 M Tris
HCl, pH 7.50 1M NaCl. The pH of the culture medium was adjusted to
pH 7.4 before applying to the pre-equilibrated column. The column
was washed with 500 ml of equilibration buffer and followed by a
linear salt gradient from 0.1 to 1.1 M NaCl. Fractions (7 ml total)
were assayed for lactoferrin content and purity using SDS/PAGE and
silver staining. Fractions containing LF were dialyzed against
0.025 M Tris HCl, pH 7.5/0.1M NaCl and lyophilized.
EXAMPLE 7
Quantitation of Human LF
[0098] Recombinant lactoferrin was quantitated using an ELUSA assay
essentially as described by Vilja et al., J. Immunol. Methods,
76:73-83 (1985). A sensitivity of 5 ng of lactoferrin was obtained
using the non-competitive Avidin-biotin assay. Human LF isolated
from breast milk (Sigma) was used as standard. Biotinylated human
lactoferrin IgG was obtained from Jackson Immunoresearch
laboratories, West Grove, Pa.
EXAMPLE 8
N-Terminal Sequencing
[0099] Five .mu.g of purified recombinant human LF was resolved on
an SDS-polyacrylamide gel and transferred to Problott, a
polyvinylidene difluride-type membrane, following manufacturers
instructions (Applied Biosystems). Human LF was detected with
Comassie Brilliant Blue staining and destained. This human LF band
was excised, washed thoroughly with distilled H.sub.2O and
air-dried. The N-terminal amino acid sequence of the first ten
amino acids of human LF was determined by the automated Edman
degradation procedure using an applied Biosystems Pulsed-liquid
phase sequencer (Model 477A).
[0100] With reference to FIG. 8, panel A illustrates a Silver
stained SDS-polyacrylamide gel analysis of recombinant human LF
secretion and purification. Lane 1 contains breast milk human LF
standard (500 ng). Lanes 2 and 3 contain samples of the growth
medium (40 .mu.g) from induced control AO7 and transformant #1
respectively. Lanes 4-8 contain 100 .mu.l aliquots of eluted
fractions (#25, 30, 35, 40, and 45 respectively) collected from the
CM-sephadex purification of recombinant LF from the growth medium
of transformant #1. The position of the molecular weight markers
(BioRad Richmond, Calif.) are indicated on the left. Sizes are
given in kilo Daltons. Panel B illustrates a Western immunoblot
analysis of duplicate samples as described in panel A using a
specific polyclonal antibody directed against human LF with
detection with .sup.125I-protein A. Panel C illustrates #6
N-terminal amino acid sequence of recombinant human LF. Recombinant
human LF was sequenced from the N-terminus through 10 residues and
is identical to breast milk human LF with the exception of the
additional alanine generated in our construction to provide the
.alpha.-amylase signal sequence cleavage site.
EXAMPLE 9
Deglycosylation
[0101] Deglycosylation was performed using N-glycosidase F
(Boehringer Mannheim). A. oiyzae growth medium containing 0.5 .mu.g
lactoferrin was denatured for 3 minutes at 100.degree. C. in the
presence of 0.01 % SDS. Standard LF from human milk was treated
similarly. The samples were subsequently placed on ice for five
minutes. N-glycosidase F reactions were conducted in 0.4 M sodium
phosphate, (pH 6.8); 0.08% Triton; 0.1% .beta.-mercaptoethanol and
1 unit of enzyme and incubated at 37.degree. C. for sixteen hours.
PAGE and Western analysis was performed using an IgG specifically
directed against human lactoferrin to detect an increase in
mobility of digested samples.
[0102] With reference to FIG. 9, recombinant human LF was
characterized. Panel A illustrates the deglycosylation of
lactoferrin. Western analysis of glycosylated and deglycosylated
lactoferrin using a specific polyclonal antibody was directed
against human lactoferrin with detection with .sup.125I-protein A.
The first panel contains authentic breast milk human LF (500 ng)
untreated (-) and treated (+) with N-glycosidase F. The second
panel contains purified recombinant human LF (500 ng) untreated (-)
and treated (+) with N-glycosidase F. The size of glycosylated
human LF is indicated with the arrow. Panel B illustrates a
functional analysis of recombinant lactoferrin with regard to
iron-binding capacity. Panel A and B show the .sup.59Fe filter
binding assay of duplicate samples of authentic breast milk human
LF and purified recombinant human LF, respectively, at the
concentrations indicated. The first lane in both panels contain BSA
(5 .mu.g) as a negative control.
[0103] Lactoferrin contains two N-acetyllactamine type glycans
attached through N-glycosidic linkages. To determine if recombinant
lactoferrin was glycosylated correctly, the protein was treated
with N-glycosidase F, resolved on SDS-polyacrylamide
electrophoresis, transferred to nitrocellulose and probed using a
specific IgG directed against human lactoferrin (FIG. 11A).
N-glycosidase F hydrolyses at the glycosylamine linkage generating
a carbohydrate free peptide of smaller molecular weight. Comparison
of recombinant LF with purified LF from human milk, illustrates
that both proteins co-migrate upon digestion with N-glycosidase F
suggesting that the recombinant protein has a glycosylation pattern
similar to native LF.
[0104] Lactoferrin has a bilobal structure with each lobe having
the capacity to bind tightly, but reversibly, one Fe.sup.3+ ion.
The iron-binding properties of lactoferrin are crucial for its
functional roles. To test if recombinant human LF expressed and
secreted in A. oryzae has an iron binding capacity similar to
authentic lactoferrin, an .sup.59Fe micro filter binding assay was
developed. Purified human lactoferrin isolated from the growth
medium of transformant #1 was dialyzed against 0.1M citric acid (pH
2.0) to generate apo-human LF. Native lactoferrin from human milk
was treated similarly. Excess .sup.59Fe (0.2 mCi) was added to
these samples in an equal volume of 1 M bicarbonate, followed by
incubation at 37.degree. C. for 30 minutes. Samples were applied to
nitrocellulose membrane and washed several times with bicarbonate.
The filter was visualized by autoradiography and Fe-binding was
quantitated using a betagon blot analyzer. As illustrated in FIG.
11B, both recombinant and native LF showed a similar level of iron
binding at all concentrations tested. The results demonstrate that
recombinant human LF is indistinguishable from native human LF in
its capacity to bind iron.
[0105] With reference to FIG. 2, the complete cDNA sequence for
human lactoferrin protein is depicted. The cDNA coding for
lactoferrin is used to create plasmids and transform eucaryotic
cells and to produce the lactoferrin protein.
[0106] Strains of Aspergillus used in the present invention are
auxotrophic mutants that contain a defective pry4 gene that results
in an inability to synthesis orotidine 5' phosphate (OMP)
decarboxylase. The enzyme is required for uridine synthesis. The
strain cannot grow on media lacking uridine. The plasmid contains a
selectable marker, i.e., a sequence that encodes the gene for OMP
decarboxylase. Uptake of the plasmid by the Aspergillus can
therefore be selected for by growth on media lacking uridine. The
Aspergillus is transformed by the plasmid such that it can grow on
the uridine deficient media.
EXAMPLE 10
Expression of the 3' Iron-Binding Domain of Human Lactoferrin-E.
Coli
[0107] The 3' iron-binding domain of human lactoferrin (hLF) was
expressed in Escherichia coli using the bacterial expression
plasmid, PT7-7 as described by Tabor, S. and Richardson, C., Proc.
Natl. Acad. Sci. U.S.A., 82:1074-1078 (1985). pGEMhLFc, containing
the cDNA for the complete hLF cDNA (Ward, P. P., et al. Gene.
122:219-223 (1992)), was digested with Sma I and Hind III to
release a 1.5 kb fragment encoding the 3' iron-binding domain of
hLF. This 1.5 kb Sma I/Hind III fragment was subcloned in-frame
into Sma I/Hind II digested PT7-7, under the control of the strong
inducible T7 promoter, generating PT7-7hLF3'.
[0108] PT7-7hLF3' was transformed into a protease deficient strain
of E.coli which had previously been transformed with pGP1-2 plasmid
which contained the T7 polymerase under the control of the
.lambda.pL promoter as described by Conneely, O. M., et al. In:
Hormone Action and Molecular Endocrinology. 5-48-5-50 (1989)). The
PT7-7 plasmid contained an ampicillin resistance gene while the
pGP1-2 plasmid contained a kanamycin resistant gene allowing dual
antibiotic resistance selection for transformants containing both
plasmids. Transformants obtained were cultured overnight in LB
broth containing ampicillin (50 .mu.g/ml) and kanamycin (50
.mu.g/ml) at 30.degree. C./250 rpm. Overnight cultures were
subcultured into LB (500 ml) containing ampicillin and kanamycin
and grown at 30.degree. C./250 rpm until an O.D..sub.600nm of
0.5-0.6 was obtained. At 30.degree. C. the .lambda. repressor bound
to the .lambda.pL promoter, thus blocking T7 polymerase production.
Induction of the recombinant protein was achieved by raising the
temperature to 42.degree. C. for one hour to inactivate the
.lambda. repressor thus allowing T7 polymerase production. The
temperature was lowered to 30.degree. C. for a further two hours,
turning off .lambda.pL directed transcription and allowing the
production of the recombinant protein as the T7 polymerase bound to
the T7 promoter to specifically induce expression of the
recombinant lactoferrin 3' iron-binding domain.
[0109] Western Immunoblot analysis was performed to determine if
the 3' iron binding domain was expressed in the bacterial cells
under the control of the T7 promoter and to monitor its
purification. The cells were harvested at 5000 g and resuspended in
15 ml of PBS (pH 7.4). Total cellular extracts were prepared by
sonication for 1 minute on ice. The sonicate was centrifuged at
13,000 g for 40 minutes at 4.degree. C. The supernatant was removed
and the pellet was resuspended in 50 ml of denaturation buffer (5M
urea, 2% triton, 5 mM EDTA, 0.01% Tween 20, 50 mM TrisCl, pH 7.5)
and centrifuged at 48,000 g for one hour. The supernatant
containing the soluble fraction was recovered. Protein
concentration was determined using the Bradford reagent according
to manufacturers instructions (BioRad, Richmond, Calif.). Protein
samples (40 .mu.g) were resolved by SDS-PAGE and transferred to a
nitrocellulose filter electrophorectically using the Western
Immunoblot procedure. The filter was blocked with Tris-buffered
saline (TBS, 0.05 M Tris/0.15 M NaCl, pH 7.5) containing 2% dried
milk, and then incubated for 2 hours in the same with the addition
of a specific polyclonal IgG (1 .mu.g/ml) directed against hLF
(Sigma, St. Louis, Mo.). The filter was washed (5.times.10 min) in
TBS/0.05% Nonidet P40 followed by incubation with 5 .mu.Ci of
.sup.125I protein A in TBS/2% dried milk. The filter was washed
(5.times.10 min) in TBS/0.05 % Nonidet P40, dried and exposed
overnight in Kodak XAR5 film at -70.degree. C. The film was
developed by autoradiography.
[0110] The results of the Western analysis are shown in FIG. 10. An
immunoreactive band at the expected size (50 kDa) for the hLF 3'
iron-binding domain was evident in the cellular extract from
induced cells and was absent in control uninduced cells (FIG. 10,
lanes 1 and 2). The hLF 3' iron-binding domain associates with the
cellular homogenate insoluble fraction (FIG. 10, lane 3) and hence
required a further solubilization step in a denaturation buffer to
prepare the hLF in a soluble form (FIG. 10, lane 4).
[0111] Analysis of a coomassie-stained SDS-PAGE gel also showed the
presence of a 50 kDa protein in the cellular extract from induced
cultures which was absent in control uninduced cultures (FIG. 11,
lanes 2 and 3). The recombinant protein was expressed at levels up
to 10 mg/l and represented approximately 5% of the total cellular
protein. The hLF 3' iron-binding domain did not associate with the
soluble homogenate fraction (FIG. 11, lane 4) and hence required a
further solubilization step in a denaturation buffer to prepare the
hLF in a soluble form (FIG. 11, lane 5). Purification and
solubilization of the recombinant hLF 3' iron-binding domain
resulted in a 50% yield of recoverable protein and represented the
major protein band in this fraction.
[0112] In summary, we have successfully produced recombinant hLF 3'
iron-binding domain in E.coli under the control of the strong
inducible T7 promoter. The recombinant protein was expressed and
purified in a soluble form from the cellular extracts at levels up
to 5 mg/l.
EXAMPLE 12
Expression and Purification of an N-Terminal Lactoferrin Fragment
(AA 1-52) in Escherichia Coli
[0113] An N-terminal human lactoferrin fragment (AA 1-52), encoding
the bactericidal domain of hLF, reported by Bellamy et al., supra,
was expressed and purified from E. coli. The bovine lactoferrin
fragment also reported by Bellamy, et al. is produced by the same
method illustrated here for the human fragment. This was achieved
using the glutathione S-transferase (GST) Gene Fusion System
(Pharmacia, Piscataway, N.J.) where the lactoferrin fragment was
expressed as a fusion protein with glutathione S-transferase
[Smith, D. S., et al., Gene, 67:31-40 (1988)] and a protease
cleavage site allowing production of the bactoricidal domain by
cleavage from GST.
[0114] A 156 bp human lactoferrin fragment encoding AA 1-52,
containing Sma I/BamH I ends was obtained by polymerase chain
reaction (PCR) amplification of pGEMhLFc plasmid DNA [Ward, P. P.,
et al., Biotechnology, 10:784-789 (1992)]. The oligonucleotide
primers used were as follows: 5' end oligonucleotide as shown in
SEQ. ID. NO. 11 CTGCCCGGGCGTAGGAGAAGGAGTGTT 3' end oligonucleotide
as shown in SEQ. ID. No. 12 CATGGATCCTGTTTTACGCAATGGCCTGGATACA
[0115] This PCR fragment was digested with Sma I and BamH I and
repaired using the Klenow Fragment of DNA polymerase I. This
fragment was subcloned into BamH I repaired pGEX-3X generating
pGEX-3XLFN-1. This fused the lactoferrin cDNA fragment in frame,
downstream from the glutathione S-transferase gene and under the
control of the strong, inducible tac promoter. All PCR amplified
products and construction junctions were sequenced using the
commercially available Sequenase version 2.0 kit (United states
Biochemical Corp, Cleveland, Ohio).
[0116] pGEX-3XLFN-1 was transformed into the bacterial strain,
JM109. Transformants obtained were cultured overnight in LB (50 ml)
containing ampicillin (50g/ml) at 37.degree. C./250 rpm. Overnight
cultures were subcultured into LB (500 ml) containing ampicillin
(50 g/ml) and grown at 37.degree. C./250 rpm until an 0D.sub.600nm
of 0.6-0.8 was obtained. Isopropyl-D-thiogalactopyranoside (IPTG)
was added to the culture medium at a concentration of 1 mM to turn
on the tac promoter resulting in expression of the glutathione
S-transferase/LFN-1 fusion protein. Growth under these conditions
continued for 4 hours after which the cells were harvested at 5,000
g and resuspended in 5 ml of MTPBS (150 mM NaCl, 16 mM
Na.sub.2HPO.sub.4, 4 mM NaH.sub.2PO.sub.4, 1% Triton X-100, pH
7.3). Total cellular extracts were prepared by 3.times.1 minute
freeze/thaw cycles followed by mild sonication for 2.times.1
minute. The sonicate was centrifuged at 13,000 g for 20 minutes and
the supernatant obtained was applied to a glutathione sepharose 4B
column following manufacturer's instructions (Pharmacia,
Piscataway, N.J.). The glutathione S-transferase/LFN-1 fusion
protein was eluted from the column using 10 ml of elution buffer
(10 mM glutathione, 50 mM Tris pH 8.0). Fractions of 1.5 ml were
collected and dialyzed overnight against 50 mM Tris, 15% glycerol
pH 8.0.
[0117] Samples from the solubilized extracts and the purification
fractions were analyzed by SDS/PAGE followed by silver-staining.
The results of this analysis are shown in FIG. 12. A band at the
expected size (32 kDa) for the glutathione S-transferase/LFN-1
fusion protein was detected in the solubilized protein extracts
from induced JM109 cultures transformed with pGEX-3X/LFN-1 and was
absent in uninduced cultures (FIG. 12A, lanes 2 and 3). This band
migrates at a higher mobility than control induced JM109 cultures
transformed with pGEX-3X alone (FIG. 12A, lane 1). The fusion
protein was successfully purified to homogeneity over a glutathione
sepharose 4B column (FIG. 12B, lanes 1 and 2). Protein
concentration determination using the Bradford reagent (BioRad,
Richmond, Calif.) showed that the glutathione S-transferase/LFNI
fusion protein was purified at levels up to 5 mg/l. The GST fusion
protein has a protease cleavage site for the protease Kex II
between GST and the 52 amino acid protein.
[0118] In summary, a human lactoferrin fragment, encoding a
bactericidal domain of this protein, has been successfully
expressed as a fusion protein with glutathione S-transferase an E.
coli expression system. This fusion protein was purified to
homogeneity at levels up to 5 mg/l. The bactericidal protein is
obtained by cleavage with the protease Kex II to cleave the GST
portion from the bactericidal domain.
EXAMPLE 13
Expression of Bovine and Porcine Lactoferrin in Aspergillus
Oryzae
[0119] A universal A. Oryzae expression vector is constructed to
allow in frame subcloning of any cloned cDNA of interest. This
vector, pAG, is similar to the vector pAhLFG(+1) utilized for the
expression of human lactoferrin in A. Oryzae above. A 680 bp
.alpha.-amylase fragment encoding the promoter, signal sequence and
the alanine residue from the start of the mature -amylase II gene,
is obtained by polymerase chain reaction (PCR) amplification of
pAhLFG(+1). The oligonucleotide primers are as follows: 5' end
oligonucleotide, SEQ. ID. NO. 13 5.degree.
CGGAATTCATGGTGTTTGATCATIT 3' end oligonucleotide, SEQ. ID. NO. 14
5'TGGAATTCGATCGCGGATCCGCAATGCATGCAGCCAAAGCAGGTGCCG CGAC
[0120] The 5' end oligonucleotide encodes an EcoR I site and the 3'
end oligonucleotide contains an Nsi I site, flanked by a BamH I
site. This amplified DNA is digested with EcoR I and BamH I and
subcloned into EcoR I/BamH I digested pAhLFG(+1) generating pAG.
All PCR amplified products and construction junctions are sequenced
using the commercially available Sequenase version 2.0 kit (United
States Biochemical Corp., Cleveland, Ohio).
[0121] A schematic representation of this expression plasmid is
outlined in FIG. 13. Restriction enzyme digestion of this
expression plasmid with Nsi I, followed by repair using DNA
polymerase I allows subcloning of any cDNA of interest in frame
with the .alpha.-amylase signal sequence and alanine residue from
the start of the mature .alpha.-amylase II gene. 5' and 3'
oligonucleotide primers are designed to contain Acc 1 ends, and
used to obtain the full length cDNA encoding for mature porcine and
bovine lactoferrin using polymerase chain reaction (PCR)
amplification of their known DNA sequence. The PCR fragment thus
obtained is digested with Acc I and repaired using the Klenow
fragment of DNA polymerase I for in frame subcloning into Nsi I
blunt-ended pAG. The plasmids are then be transformed into the
pyrG- strain of A. Oryzae to obtain expression and secretion of
these cDNAs as previously described for human lactoferrin.
EXAMPLE 14
Expression of Human Lactoferrin in Saccharomyces Cerevisiae
[0122] The complete human lactoferrin (hLF) cDNA was expressed in
Saccharomyces cerevisiae using the yeast expression plasmid, YEP
[McDonnell, D. P. et al., J. Steroid Biochem, Molec. Biol.,
39:291-297 (1991)]. A 2.2 kb fragment encoding the complete hLF
cDNA SEQ. ID No. 1 was generated using the polymerase chain
reaction. This fragment contained and XhoI restriction enzyme site
at its 5' end and an Asp718 restriction enzyme site at its 3' end.
The 2.2 kb fragment was subcloned, in frame, into XhoI/Asp718
digested YEP to yield, YEPLFc.
[0123] Transcription of the hLF cDNA was under the control of the
copper responsive yeast metallothionein promoter (CUP1). hLF was
produced as a ubiquitin fusion protein. The fusion protein is short
lived in the yeast cells and is processed to produce unfused
protein upon folding.
[0124] YEPLFc was transformed into a protease deficient strain of
S.cerevisiae, by standard techniques [Ito, H., et al., J.
Bacteriol., 153:163-186 (1983).] This strain cannot grow unless the
growth medium is supplemented with adenine, uracil and tryptophan.
The YEP plasmid contains a tryptophan selectable marker, thus,
transformants were selected by tryptophan auxotrophy.
[0125] Transformants obtained were cultured overnight in selective
medium containing 2% glucose, 0.1% casamino acids, 0.67% yeast
nitrogen base, 0.001% adenine and 0.002% uracil at 30.degree.
C./200 rpm. When the cells reached an 0D.sub.600nm of 1.0,
1.times.10.sup.6 cells were inoculated into 10 ml of the selective
medium and 100 .mu.m CuS0.sub.4 added. The cells were grown for 24
hours at 30.degree. C./200 rpm. The purpose of adding the
CuS0.sub.4 was to induce expression of the hLF cDNA from the copper
inducible CUP1 promoter.
[0126] Western immunoblot analysis was performed to determine if
hLF was expressed in the yeast cells under the control of the CUP1
promoter. The cells were harvested by centrifugation at
5000.times.g for 5 min. and resuspended in 1 ml of Z buffer (120 mM
Na.sub.2HP0.sub.47H.sub.2O, 40 mM NaH.sub.2PO.sub.4H.sub.2O, 10 mM
KCl, 1 mM MgSO.sub.47H.sub.2O, 0.27% 2-mercaptoehanol, pH 7.0).
Total cellular extracts were prepared by glass bead homogenization.
This procedure involved mixing the yeast cells with an equal volume
of glass beads (0.5 mm, B.Braun Instruments) and vortexing for
5.times.1 min. The homogenate was centrifuged at 13,000 g for 10
min. and the supernatant removed. The protein concentration was
determined using the Bradford reagent in accordance with the
manufacturer's instructions (BioRad, Richmond, Calif.). Protein
samples (50 .mu.g) were resolved by SDS-PAGE and
electrophoretically transferred, overnight, to a nitrocellulose
filter using the western immunoblot procedure. The filter was
blocked with tris-buffered saline (TBS=0.05M Tris/0.15M NaCl, pH
7.5) containing 1% dried milk and then incubated overnight, in the
same, with the addition of a specific rabbit polyclonal antibody (1
.mu.g/ml) directed against hLF (Signa, St. Louis, Mo.). The filter
was washed in TBS/0.1% Tween 20 (5.times.5 min.) followed by
incubation with horseradish peroxidase (Amersham, UK) for 1 hour.
The filter was washed in TBS/0.3% Tween 20 (3.times.5 min.) and
then TBS/0.1% Tween 20 (3.times.5 min.). The filter was then
treated with luminol and enhancer (Amersham, UK) for 1 min., dried
and exposed for 1 min. to X-ray film. The film was developed by
autoradiography.
[0127] These data demonstrate successful production of recombinant
hLF in S. cerevisiae under the control of the copper inducible
(CUP1) promoter.
[0128] The results of the western analysis are shown in FIG. 16. An
immunoreactive band at the expected size (78 kDa) for hLF was
evident in the cellular extract from transformed S. Cerevisiae
cells. FIG. 16, lane 1.
EXAMPLE 15
Expression of hLF in Aspergillis Nidulans
[0129] Construction of the Aspergillis Nidulans Expression
Plasmid.
[0130] The plasmid used for expression of hLF cDNA is shown
schematically in FIG. 17. The cDNA of SEQ. ID No. 1 as a 2.3-kb
clone contained the secretory signal sequence and complete
translation frame. The sequence of the entire cDNA was confirmed by
dideoxy sequence analysis (Sequenase version 2.0, U.S. Biochemical,
Cleveland, Ohio). The cDNA was repaired using the Pollk and
subcloned into AccI-digested and blunt-ended pGEM4. The plasmid,
pGEMhLF, was digested with HindIII+Asp718 and repaired using Polk.
The resulting 2.3-kb hLF fragment was subcloned into a unique SmaI
site located in the multiple cloning cassette of pAL3 downstream
from the alcA promoter, Waring, R. B., et al., Gene, 79, 119-130
(1989), generating pAL3hLF. The .beta.-tubulin transcription
terminator fragment was obtained by digesting the 3'-untranslated
region of the bena gene (nt 2569-2665; May et al., 1987) with
Xbal+NheI and subcloned into XbaI-digested pAL3hLF generating
pAL3hLFT. This plasmid was used to transform A. nidulans strain GR5
(pyrG89; wa3; pyroA4)
[0131] The A.nidulans expression plasmid, pAL3hLFT, contains 300 bp
of 55'-flanking sequence of the A. nidulans alcA gene containing
all the regulatory elements necessary for controlled gene
expression. To construct pALhLFT, a 2.3-kb hLF cDNA fragment
containing 17 nucleotides of 5'-UTR, the complete hLF ORF encoding
the secretory signal peptide and mature hLF, followed by 209 nt of
3' UTR was subcloned into a unique Smal site in pAL3 downstream
from the alcA promoter. A 96-bp terminator fragment from the A.
nidulans .beta.-tubulin-encoding (benA) gene was subcloned into a
unique XbaI site downstream from the hLF cDNA sequence. The plasmid
also contains an Ap.sup.R maker and the N. crassa pyr4 selectable
marker (Waring et al., supra, 1989).
[0132] Transformation and Southern Analysis
[0133] Transformation was carried out as described by May et al.,
J. Cell Beol., 109, 2267-2274 (1989). Protoplasts were transformed
with 3 .mu.g of the expression plasmid with an efficiency of 40
transformants/.mu.g DNA. Transformats obtained were purified three
times through conidial spores. Southern blot analysis was performed
to confirm that transformants contained integrated plasmid with hLF
cDNA. A hLF-specific radiolabelled band was detected at the
expected size (2.3 kb) in lanes 1-10 but not in DNA from control
spores. These results demonstrate that hLF cDNA was integrated into
the genome of all A. nidulans transformants tested and varied
randomly from one copy (transformants Nos. 3, 6 and 10) to 20
copies (No. 5) per cell. The site of integration of the plasmid
into the A. nidulans genome is random due to the absence of
homologous sequences to target the vector into a particular
site.
[0134] Southern blot analysis was conducted of transformed A.
nidulans. Genomic DNA was isolated from ten individual A. nidulans
(GR5) transformats and untransformed spores as described by
Rasmussen, C. D. et al., J. Biol. Chem., 265, 13767-13775 (1990).
The DNA (1 .mu.g) was digested with EcoRI, size fractionated on a
0.8% agarose gel and transferred to a nitrocellulose filter and
hybridized with a radiolabelled hLF cDNA probe (2.1-kb). A sample
(20 ng) of hLF cDNA was used as a positive control (hLF cDNA).
Prehybridization and hybridization of the filter was performed in
6.times.SSC/0.1% SDS/0.5% dried milk at 65.degree. C. for 16h. The
hybridization solution contained 200 ng of .sup.32P probe (2.1 kb;
specific activity 4.times.10.sup.8 cpm/.mu.g of DNA). Filters were
washed in 2.times.SSC/0.5% SDS at 68.degree. C. for 30 min followed
by 0.5.times.SSC/0.5% SDS at 68.degree. C. for 30 min. The filter
was dried and exposed to Kodak X-AR5 film at -70.degree. C. for 30
min and developed by autoadiograpy. The autoradiography showed an
intense 2.1 kb band for hLF.
[0135] Production of hLF in Aspergillus Nidulans
[0136] Conidia (1.times.10.sup.6/ml) were cultured in minimal media
utilizing 100 mM Na acetate pH 6.5 as carbon source with or without
addition of 1.2% ethanol to induce transcription of the hLF cDNA.
GR5 was cultured as above except for the addition of 5 mM uridine
and 10 mM uracil. Media and mycelia were harvested and separated
using Miracloth (Calbiochem, San Diego, Calif). Mycelia (200 mg)
were freeze-dried and lyophilized overnight. Total cellular
extracts were prepared by homogenization in a glass teflon
homogenizer using 1 ml of phosphate-buffered saline (PBS; 137 mM
NaCl/2.7 mM KCl/4.3 mM Na.sub.2HPO.sub.47H.sub.2O/1.4 mM
K.sub.2HPO.sub.4pH 7.4) in the presence of
phenylmethylsulfonylfluorride (PMSF, 10 .mu.g). The homogenate was
centrifuged at 12000.times.g for 30 min at 4.degree. C. and the
supernatant containing the soluble fraction was recovered. The
growth medium was concentrated by freeze drying and lyophilization
and resuspended in 1/30 vol. in PBS pH 7.4. Protein concentration
was determined using the Bradford reagent according to
manufacturer's instructions (BioRad, Richmond, Calif.).
Concentrated media samples containing 40 .mu.g protein and soluble
extracts (50 .mu.g protein) were subjected to 0.1% SDS/7% PAGE,
Laenmnli, U. K., Nature, 227, 680-685 (1970). Purified lactoferrin
(250 ng, Sigma, St. Louis, Mo.) was used as standard (hLF std). The
resolved proteins were transferred to nitrocellulose filters
electrophoretically using the Western blot procedure, Towbin, H.,
et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979). Filters
were blocked with Tris-buffered saline (TBS, 0.05 M Tris/0.15 M
NaCl pH 7.5) containing 2% dried milk and then incubated by 2 h in
the same with the addition of a 1 .mu.g/ml of a specific polyclonal
IgG directed against hLF (Sigma, St. Louis, Mo.). Filter washes
(5.times.10 min) were in TBS/0.05% Nonidet P40 followed by
incubation with 1 .mu.Ci of [.sup.125I] protein A in BS/2% dried
milk. The filter was washed (5.times.10 min) with TBS/0.05% Nonidet
P-40, dried and exposed overnight to Kodak XAR5 film at -70.degree.
C. The film was then developed by autoradiography. The
autoradiographs demonstrate production of hLF. Western analysis was
performed to determine if the hLF cDNA was expressed in the A.
nidulans transformats under the control of the alcA promoter.
[0137] Conidia (1.times.10.sup.6/ml) from transformat No. 5, which
contained the highest number of copies of integrated hLF cDNAs, and
from untransformed GR5 were inoculated into minimal medium
utilizing glucose as the carbon source. After 18 h, the cultures
were harvested, washed and reinoculated into minimal medium
supplemented with 1.2% ethanol and grown for an additional 12 or 24
h before harvesting the cultures. Cell extracts and samples of the
growth medium were resolved by SDS-PAGE, transferred to
nitrocellulose and immunoblotted using a specific polyclonal IgG
directed against hLF. An immunoreactive band indistinguishable from
native hLF was evident in the cells and growth medium from
transformat No. 5 after 12 and 24 h growth only after ethanol
induction. Cell extracts or growth medium obtained from
untransformed GR5 did not contain an immunoreactive band even after
addition of ethanol. These results demonstrate that hLF is
expressed in transformed A. nidulans under the control of the alcA
promoter.
[0138] Western analysis revealed hLF in the cells in all of the
remaining transformants. In general there was a correlation between
the plasmid copy number and the expression levels obtained. In the
medium hLF was detected only with transformats containing multiple
copies of integrated expressed plasmid (Nos. 1, 5, 7 and 10).
[0139] In order to monitor the levels of hLF produced in the
system, a pilot fermentation of transformant No. 5 was carried out
using the growth parameters described above. ELISA analysis, Vilja,
P., et al., J. Immunol. Methods, 76, 73-83 (1985), using a specific
biotinylated IgG directed against hLF demonstrated that the total
level of recombinant hLF produced was 5 .mu.g/ml with approx. 30%
(1.5-2.0 .mu.g/ml) of this material secreted into the medium.
[0140] Iron Binding Analysis of hLF.
[0141] To test if recombinant lactoferrin synthesized and secreted
in A. nidulans has an iron binding capacity similar to authentic
human lactoferrin, samples of the growth medium of transformant No.
5 and untransformed GR5 spores were examined using an .sup.59Fe
microfilter-binding assay to detect .sup.59Fe-bound lactoferrin.
Iron-binding (.sup.59Fe) is detected in the medium from
transformant No. 5 but not in the medium from control untransformed
GR5 spores. These results indicate that hLF produced in A. nidulans
is biologically active in its capacity to bind .sup.59Fe.
[0142] The data demonstrate the successful production of
biologically active hLF in A. nidulans. The levels of hLF produced
in A. nidulans were approx. 5 .mu.g/ml with 30% of the hFL secreted
into the growth medium. The secreted hLF was identical to native
breast milk LF with regard to size and immunoreactivity.
Furthermore, the hLF was capable of binding iron. Although hLF has
been reported to contain anti-fungal properties, neither the re-hLF
nor native hLF when added to the growth medium, retarded the growth
of this strain of A. nidulans. The production of biologically
active hLF in A. nidulans will facilitate testing of possible
nutritional and therapeutic uses of this protein.
EXAMPLE 16
Production of DNA Sequence Substitution Analogs
[0143] FIG. 18 shows the restriction enzyme cleavage sites in the
SEQ I. D. No. 1 cDNA for cleavage by various endonucleases. Table 2
lists the alternative codons that code for the 20 common amino
acids. DNA sequence substitution analogs that also code for human
lactoferrin can be constructed by choosing alternate codons from
Table 2.to alter the DNA Sequence between a pair of cleavage sites
selected from FIG. 18. Alternative codons are assembled into a
synthetic oligonucleotide by conventional methods and the synthetic
oligo is substituted into the endonuclease treated DNA of Sequence
ID. No. 1 by the methods described in "Molecular Cloning. A
Laboratory Manual", 2d Edition, Cold Spring Harbor Laboratory Press
(1989), to produce a substitution analog. Other methods generally
known to those skilled in the art can also be employed to obtain
substitution analogs of DNA sequences. The alteration of the DNA by
cleavage and codon substitution maybe repeated to substitute
substantial portions of the original DNA sequence with alternative
codons without altering the protein expressed by the DNA of
Sequence ID. No. 1. The same methods can of course be used to make
substitution analogs of the cDNA of SEQ ID No. 3 and 5. Alteration
of a DNA sequence which produces no change in the protein expressed
by the DNA sequence might, for example, be conducted to increase
protein expression in a particular host cell by increasing the
occurrence of codons that correspond to amino acid tRNAs found in
higher concentration in the host cell. Such altered DNA sequences
for substitution analogs can be easily produced by those of
ordinary skill in the art following the method set out above, or
other alternative techniques for altering the DNA sequence while
obtaining the same protein on expression. Substitution analogs can
be obtained by substitution of oligonucleotides at restriction
cleavage sites as described above, or by other equivalent methods
that change the codons while preserving the amino acid sequence of
the expressed protein.
6 TABLE 2 AMINO ACID CODONS Phe TTT TCC Leu TTA TTG CTT CTC CTA CTG
Ile ATT ATC ATA Met ATG Val GTT GTC GTA GTG Ser TCT TCC TCA TCG AGT
AGC Pro CCT CCC CCA CCG Thr ACT ACC ACA ACG Ala GCT GCC GCA GCG Tyr
TAT TAC Gly GGT GGC GGA GGG His CAT CAC Gln CAA CAG Asn AT AAC Lys
AAA AAG Asp GAT GAC Glu GAA GAG Cys TGT TGC Trp TGG Arg CGT CGC CGA
CGG AGA AGG TERMINATION TAA SIGNALS TAG TGA
[0144] In conclusion, it is seen that the present invention and the
embodiments disclosed herein are well adapted to carry out the
objectives and obtain the end set forth in this application.
Certain changes can be made in the method and apparatus without
parting from the spirit and scopes of this invention. It is
realized that changes are possible and that it is further intended
that each element or step presided in any of the filing claims is
to be understood as to referring to all equivalent elements or
steps for accomplishing the essentially the same results in
substantially the same or equivalent manner. It is intended to
cover the invention broadly in whatever form its principles may be
utilized. The present invention, therefore, is well adapted to
carry out the objects and obtain the ends and advantages mentioned,
as well as others inherent therein.
Sequence CWU 1
1
14 1 2360 DNA Homo sapiens 1 gaattccgac cgcagacatg aaacttgtct
tcctcgtcct gctgttcctc ggggccctcg 60 gactgtgtct ggctggccgt
aggagaagga gtgttcagtg gtgcaccgta tcccaacccg 120 aggccacaaa
atgcttccaa tggcaaagga atatgagaag agtgcgtggc cctcctgtca 180
gctgcataaa gagagactcc cccatccagt gtatccaggc cattgcggaa aacagggccg
240 atgctgtgac ccttgatggt ggtttcatat acgaggcagg cctggccccc
tacaaactgc 300 gacctgtagc ggcggaagtc tacgggaccg aaagacagcc
acgaactcac tattatgccg 360 tggctgtggt gaagaagggc ggcagctttc
agctgaacga actgcaaggt ctgaagtcct 420 gccacacagg ccttcgcagg
accgctggat ggaatgtgcc tatagggaca cttcgtccat 480 tcttgaattg
gacgggtcca cctgagccca ttgaggcagc tgtggccagg ttcttctcag 540
ccagctgtgt tcccggtgca gataaaggac agttccccaa cctgtgtcgc ctgtgtgcgg
600 ggacagggga aaacaaatgt gccttctcct cccaggaacc gtacttcagc
tactctggtg 660 ccttcaagtg tctgagagac ggggctggag acgtggcttt
tatcagagag agcacagtgt 720 ttgaggacct gtcagacgag gctgaaaggg
acgagtatga gttactctgc ccagacaaca 780 ctcggaagcc agtggacaag
ttcaaagact gccatctggc ccgggtccct tctcatgccg 840 ttgtggcacg
aagtgtgaat ggcaaggagg atgccatctg gaatcttctc cgccaggcac 900
aggaaaagtt tggaaaggac aagtcaccga aattccagct ctttggctcc cctagtgggc
960 agaaagatct gctgttcaag gactctgcca ttgggttttc gagggtgccc
ccgaggatag 1020 attctgggct gtaccttggc tccggctact tcactgccat
ccagaacttg aggaaaagtg 1080 aggaggaagt ggctgcccgg cgtgcgcggg
tcgtgtggtg tgcggtgggc gagcaggagc 1140 tgcgcaagtg taaccagtgg
agtggcttga gcgaaggcag cgtgacctgc tcctcggcct 1200 ccaccacaga
ggactgcatc gccctggtgc tgaaaggaga agctgatgcc atgagtttgg 1260
atggaggata tgtgtacact gcaggcaaat gtggtttggt gcctgtcctg gcagagaact
1320 acaaatccca acaaagcagt gaccctgatc ctaactgtgt ggatagacct
gtggaaggat 1380 atcttgctgt ggcggtggtt aggagatcag acactagcct
tacctggaac tctgtgaaag 1440 gcaagaagtc ctgccacacc gccgtggaca
ggactgcagg ctggaatatc cccatgggcc 1500 tgctcttcaa ccagacgggc
tcctgcaaat ttgatgaata tttcagtcaa agctgtgccc 1560 ctgggtctga
cccgagatct aatctctgtg ctctgtgtat tggcgacgag cagggtgaga 1620
ataagtgcgt gcccaacagc aatgagagat actacggcta cactggggct ttccggtgcc
1680 tggctgagaa tgctggagac gttgcatttg tgaaagatgt cactgtcttg
cagaacactg 1740 atggaaataa caatgaggca tgggctaagg atttgaagct
ggcagacttt gcgctgctgt 1800 gcctcgatgg caaacggaag cctgtgactg
aggctagaag ctgccatctt gccatggccc 1860 cgaatcatgc cgtggtgtct
cggatggata aggtggaacg cctgaaacag gtgctgctcc 1920 accaacaggc
taaatttggg agaaatggat ctgactgccc ggacaagttt tgcttattcc 1980
agtctgaaac caaaaacctt ctgttcaatg acaacactga gtgtctggcc agactccatg
2040 gcaaaacaac atatgaaaaa tatttgggac cacagtatgt cgcaggcatt
actaatctga 2100 aaaagtgctc aacctccccc ctcctggaag cctgtgaatt
cctcaggaag taaaaccgaa 2160 gaagatggcc cagctcccca agaaagcctc
agccattcac tgcccccagc tcttctcccc 2220 aggtgtgttg gggccttggc
tcccctgctg aaggtgggga ttgcccatcc atctgcttac 2280 aattccctgc
tgtcgtctta gcaagaagta aaatgagaaa ttttgttgaa aaaaaaaaaa 2340
aaaaaaaaaa aaaaaaaaaa 2360 2 711 PRT Homo sapiens 2 Met Lys Leu Val
Phe Leu Val Leu Leu Phe Leu Gly Ala Leu Gly Leu 1 5 10 15 Cys Leu
Ala Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Thr Val Ser 20 25 30
Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Arg 35
40 45 Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile
Gln 50 55 60 Cys Ile Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val
Thr Leu Asp 65 70 75 80 Gly Gly Phe Ile Tyr Glu Ala Gly Leu Ala Pro
Tyr Lys Leu Arg Pro 85 90 95 Val Ala Ala Glu Val Tyr Gly Thr Glu
Arg Gln Pro Arg Thr His Tyr 100 105 110 Tyr Ala Val Ala Val Val Lys
Lys Gly Gly Ser Phe Gln Leu Asn Glu 115 120 125 Leu Gln Gly Leu Lys
Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly 130 135 140 Trp Asn Val
Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly 145 150 155 160
Pro Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser 165
170 175 Cys Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg
Leu 180 185 190 Cys Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser
Gln Glu Pro 195 200 205 Tyr Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu
Arg Asp Gly Ala Gly 210 215 220 Asp Val Ala Phe Ile Arg Glu Ser Thr
Val Phe Glu Asp Leu Ser Asp 225 230 235 240 Glu Ala Glu Arg Asp Glu
Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg 245 250 255 Lys Pro Val Asp
Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser 260 265 270 His Ala
Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp 275 280 285
Asn Leu Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro 290
295 300 Lys Phe Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu
Phe 305 310 315 320 Lys Asp Ser Ala Ile Gly Phe Ser Arg Val Pro Pro
Arg Ile Asp Ser 325 330 335 Gly Leu Tyr Leu Gly Ser Gly Tyr Phe Thr
Ala Ile Gln Asn Leu Arg 340 345 350 Lys Ser Glu Glu Glu Val Ala Ala
Arg Arg Ala Arg Val Val Trp Cys 355 360 365 Ala Val Gly Glu Gln Glu
Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu 370 375 380 Ser Glu Gly Ser
Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys 385 390 395 400 Ile
Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly 405 410
415 Gly Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala
420 425 430 Glu Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn
Cys Val 435 440 445 Asp Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val
Val Arg Arg Ser 450 455 460 Asp Thr Ser Leu Thr Trp Asn Ser Val Lys
Gly Lys Lys Ser Cys His 465 470 475 480 Thr Ala Val Asp Arg Thr Ala
Gly Trp Asn Ile Pro Met Gly Leu Leu 485 490 495 Phe Asn Gln Thr Gly
Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser 500 505 510 Cys Ala Pro
Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile 515 520 525 Gly
Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg 530 535
540 Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly
545 550 555 560 Asp Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn
Thr Asp Gly 565 570 575 Asn Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys
Leu Ala Asp Phe Ala 580 585 590 Leu Leu Cys Leu Asp Gly Lys Arg Lys
Pro Val Thr Glu Ala Arg Ser 595 600 605 Cys His Leu Ala Met Ala Pro
Asn His Ala Val Val Ser Arg Met Asp 610 615 620 Lys Val Glu Arg Leu
Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe 625 630 635 640 Gly Arg
Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser 645 650 655
Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg 660
665 670 Leu His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr
Val 675 680 685 Ala Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro
Leu Leu Glu 690 695 700 Ala Cys Glu Phe Leu Arg Lys 705 710 3 2347
DNA Bos taurus 3 gagccttcgt tccggagtcg ccccaggacg ccagcccatg
aagctcttcg tccccgccct 60 cctgtccctt ggagcccttg gactgtgtct
ggctgccccg aggaaaaacg ttcgatggtg 120 taccatctcc caacctgagt
ggttcaaatg ccgcagatgg cagtggagga tgaagaagct 180 gggtgctccc
tctatcacct gtgtgaggcg ggcctttgcc ttggaatgta ttccgggcat 240
cgcggagaaa aaggcggatg ctgtgaccct ggatggtggc atggtgtttg aggcgggccg
300 ggacccctac aaactgcggc cagtagcagc agagatctat gggacgaaag
agtctcccca 360 aacccactat tatgctgtgg ccgtcgtgaa gaagggcagc
aactttcagc tggaccagct 420 gcaaggccgg aagtcctgcc atacgggcct
tggcaggtcc gctgggtgga tcatccctat 480 gggaatcctt cgcccgtact
tgagctggac agagtcactc gagcccctcc agggagctgt 540 ggctaaattc
ttctctgcca gctgtgttcc ctgcattgat agacaagcat accccaacct 600
gtgtcaactg tgcaaggggg agggggagaa ccagtgtgcc tgctcctccc gggaaccata
660 cttcggttat tctggtgcct tcaagtgtct gcaggacggg gctggagacg
tggcttttgt 720 taaagagacg acagtgtttg agaacttgcc agagaaggct
gacagggacc agtatgagct 780 tctctgcctg aacaacagtc gggcgccagt
ggatgcgttc aaggagtgcc acctggccca 840 ggtcccttct catgctgtcg
tggcccgaag tgtggatggc aaggaagact tgatctggaa 900 gcttctcagc
aaggcgcagg agaaatctgg aaaaaacaag tctcggagct tccagctctt 960
tggctctcca cccggccaga gggacctgct gttcaaagac tctgctcttg ggtttttgag
1020 gatcccctcg aaggtagatt cggcgctgta cctgggctcc cgctacttga
ccaccttgaa 1080 gaacctcagg gaaactgcgg aggaggtgaa ggcgcggtac
accagggtcg tgtggtgtgc 1140 cgtgggacct gaggagcaga agaagtgcca
gcagtggagc cagcagagcg gccagaacgt 1200 gacctgtgcc acggcgtcca
ccactgacga ctgcatcgtc ctggtgctga aaggggaagc 1260 agatgccctg
aacttggatg gaggatatat ctacactgcg ggcaagtgtg gcctggtgcc 1320
tgtcctggca gagaaccgga aatcctccaa acacagtagc ctagattgtg tgctgagacc
1380 aacggaaggg taccttgccg tggcagttgt caagaaagca aatgaggggc
tcacatggaa 1440 ttctctgaaa gacaagaagt cgtgccacac cgccgtggac
aggactgcag gctggaacat 1500 ccccatgggc ctgatcgtca accagacagg
ctcctgcgca tttgatgaat tctttagtca 1560 gagctgtgcc cctggggctg
acccgaaatc cagactctgt gccttgtgtg ctggcgatga 1620 ccagggcctg
gacaagtgtg tgcccaactc taaggagaag tactatggct ataccggggc 1680
tttcaggtgc ctggctgagg acgttgggga cgttgccttt gtgaaaaacg acacagtctg
1740 ggagaacacg aatggagaga gcactgcaga ctgggctaag aacttgaatc
gtgaggactt 1800 caggttgctc tgcctcgatg gcaccaggaa gcctgtgacg
gaggctcaga gctgccacct 1860 ggcggtggcc ccgaatcacg ctgtggtgtc
tcggagcgat agggcagcac acgtgaaaca 1920 ggtgctgctc caccagcagg
ctctgtttgg gaaaaatgga aaaaactgcc cggacaagtt 1980 ttgtttgttc
aaatctgaaa ccaaaaacct tctgttcaat gacaacactg agtgtctggc 2040
caaacttgga ggcagaccaa cgtatgaaga atatttgggg acagagtatg tcacggccat
2100 tgccaacctg aaaaaatgct caacctcccc gcttctggaa gcctgcgcct
tcctgacgag 2160 gtaaagcctg caaagaagct agcctgcctc cctgggcctc
agctcctccc tgctctcagc 2220 cccaatctcc aggcgcgagg gaccttcctc
tcccttcctg aagtcggatt tttgccaagc 2280 tcatcagtat ttacaattcc
ctgctgtcat tttagcaaga aataaaatta gaaatgctgt 2340 tgaaaaa 2347 4 708
PRT Bos taurus 4 Met Lys Leu Phe Val Pro Ala Leu Leu Ser Leu Gly
Ala Leu Gly Leu 1 5 10 15 Cys Leu Ala Ala Pro Arg Lys Asn Val Arg
Trp Cys Thr Ile Ser Gln 20 25 30 Pro Glu Trp Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu 35 40 45 Gly Ala Pro Ser Ile Thr
Cys Val Arg Arg Ala Phe Ala Leu Glu Cys 50 55 60 Ile Pro Gly Ile
Ala Glu Lys Lys Ala Asp Ala Val Thr Leu Asp Gly 65 70 75 80 Gly Met
Val Phe Glu Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val 85 90 95
Ala Ala Glu Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr 100
105 110 Ala Val Ala Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln
Leu 115 120 125 Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly Arg Ser
Ala Gly Trp 130 135 140 Ile Ile Pro Met Gly Ile Leu Arg Pro Tyr Leu
Ser Trp Thr Glu Ser 145 150 155 160 Leu Glu Pro Leu Gln Gly Ala Val
Ala Lys Phe Phe Ser Ala Ser Cys 165 170 175 Val Pro Cys Ile Asp Arg
Gln Ala Tyr Pro Asn Leu Cys Gln Leu Cys 180 185 190 Lys Gly Glu Gly
Glu Asn Gln Cys Ala Cys Ser Ser Arg Glu Pro Tyr 195 200 205 Phe Gly
Tyr Ser Gly Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp 210 215 220
Val Ala Phe Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys 225
230 235 240 Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser
Arg Ala 245 250 255 Pro Val Asp Ala Phe Lys Glu Cys His Leu Ala Gln
Val Pro Ser His 260 265 270 Ala Val Val Ala Arg Ser Val Asp Gly Lys
Glu Asp Leu Ile Trp Lys 275 280 285 Leu Leu Ser Lys Ala Gln Glu Lys
Ser Gly Lys Asn Lys Ser Arg Ser 290 295 300 Phe Gln Leu Phe Gly Ser
Pro Pro Gly Gln Arg Asp Leu Leu Phe Lys 305 310 315 320 Asp Ser Ala
Leu Gly Phe Leu Arg Ile Pro Ser Lys Val Asp Ser Ala 325 330 335 Leu
Tyr Leu Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu 340 345
350 Thr Ala Glu Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala
355 360 365 Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln
Gln Ser 370 375 380 Gly Gln Asn Val Thr Cys Ala Thr Ala Ser Thr Thr
Asp Asp Cys Ile 385 390 395 400 Val Leu Val Leu Lys Gly Glu Ala Asp
Ala Leu Asn Leu Asp Gly Gly 405 410 415 Tyr Ile Tyr Thr Ala Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu 420 425 430 Asn Arg Lys Ser Ser
Lys His Ser Ser Leu Asp Cys Val Leu Arg Pro 435 440 445 Thr Glu Gly
Tyr Leu Ala Val Ala Val Val Lys Lys Ala Asn Glu Gly 450 455 460 Leu
Thr Trp Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val 465 470
475 480 Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn
Gln 485 490 495 Thr Gly Ser Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser
Cys Ala Pro 500 505 510 Gly Ala Asp Pro Lys Ser Arg Leu Cys Ala Leu
Cys Ala Gly Asp Asp 515 520 525 Gln Gly Leu Asp Lys Cys Val Pro Asn
Ser Lys Glu Lys Tyr Tyr Gly 530 535 540 Tyr Thr Gly Ala Phe Arg Cys
Leu Ala Glu Asp Val Gly Asp Val Ala 545 550 555 560 Phe Val Lys Asn
Asp Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr 565 570 575 Ala Asp
Trp Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys 580 585 590
Leu Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu 595
600 605 Ala Val Ala Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala
Ala 610 615 620 His Val Lys Gln Val Leu Leu His Gln Gln Ala Leu Phe
Gly Lys Asn 625 630 635 640 Gly Lys Asn Cys Pro Asp Lys Phe Cys Leu
Phe Lys Ser Glu Thr Lys 645 650 655 Asn Leu Leu Phe Asn Asp Asn Thr
Glu Cys Leu Ala Lys Leu Gly Gly 660 665 670 Arg Pro Thr Tyr Glu Glu
Tyr Leu Gly Thr Glu Tyr Val Thr Ala Ile 675 680 685 Ala Asn Leu Lys
Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala 690 695 700 Phe Leu
Thr Arg 705 5 2259 DNA Sus scrofa 5 acatgaagct cttcatcccc
gccctgctgt tcctcgggac acttggactg tgtctggctg 60 cccctaagaa
aggggttcga tggtgtgtca tatccacagc agagtattca aaatgccgcc 120
agtggcaatc aaagataaga agaactaatc ccatgttctg cataaggagg gcttctccca
180 ctgactgtat ccgggccatc gcggcaaaaa gggcagatgc tgtgaccctt
gatggtggtt 240 tggtgtttga agcagaccag tacaaactgc ggccggtagc
agcggagatc tacgggacag 300 aagagaatcc ccaaacctac tattatgctg
tggctgtagt gaagaaaggt ttcaactttc 360 agaaccagct acaaggtcga
aagtcctgcc acacaggcct tggcaggtct gccgggtgga 420 atatccctat
agggttactt cgccggttct tggactgggc agggccacct gagcccctcc 480
agaaagctgt ggccaaattc ttctctcaga gctgtgtgcc ctgcgcagat ggaaatgcgt
540 atcccaacct gtgtcagctg tgcataggga aagggaaaga taaatgtgct
tgttcctccc 600 aggaaccgta ttttggctat tccggtgcct tcaactgtct
gcacaaaggg attggagatg 660 tggcttttgt caaggagagt acagtgtttg
agaacctgcc acagaaggct gaccgggaca 720 aatacgagct actctgccca
gacaatactc gaaagccagt ggaagcattc agggagtgcc 780 accttgcccg
ggtcccttct catgctgttg tggcccgaag tgtgaatggc aaggagaact 840
ccatctggga gcttctctac cagtcacaga aaaagtttgg aaaaagcaat ccacaggagt
900 tccagctctt tggctctcct ggtcagcaga aggacctcct gtttagagat
gctaccatcg 960 ggtttttgaa gatcccctca aagatagatt ctaagctgta
cctgggcctc ccgtacctta 1020 ctgccatcca gggcctgagg gaaacggcag
cggaggtgga ggcgcggcag gcgaaggtcg 1080 tgtggtgcgc cgtgggtcca
gaggagctgc gcaagtgccg gcagtggagc agccagagca 1140 gccagaacct
gaactgcagc ctggcctcca ccaccgagga ctgcatcgtc caggtgctga 1200
aaggagaagc tgatgctatg agcttggatg gaggatttat ctacactgcg ggcaagtgtg
1260 gtttggtgcc tgtcctggca gagaaccaaa aatctcgcca aagcagtagc
tcagactgtg 1320 tgcatagacc aacacaaggg tattttgccg tggcggttgt
caggaaagca aatggtggta 1380 tcacctggaa ctctgtgaga ggcacgaagt
cctgccacac tgctgtggac aggacagcag 1440 gctggaacat
ccccatgggc ctgcttgtca accagacagg ctcctgcaaa tttgacgaat 1500
tctttagtca aagctgtgct cctgggtctc agccgggatc caatctctgt gcactgtgtg
1560 ttggcaatga ccagggcgtg gacaagtgtg tgcccaacag taatgagaga
tactatggtt 1620 acaccggggc tttcaggtgc ctggctgaga atgctgggga
tgtggcgttt gtgaaagatg 1680 tcactgtctt ggacaacacg aatggacaga
acacagaaga gtgggccagg gaattgaggt 1740 cagatgactt tgagctgctg
tgccttgatg gcaccaggaa gcctgtgact gaggctcaga 1800 actgtcacct
ggctgtggcc cccagtcatg ctgtggtctc tcggaaggaa aaggcagcac 1860
aggtggaaca ggtgctactc actgagcagg ctcagtttgg aagatacgga aaagactgcc
1920 cggacaagtt ttgcttgttc cggtctgaga ccaaaaacct tctgttcaac
gacaacacgg 1980 aggttctggc ccaactccaa ggcaaaacaa catacgaaaa
atatttggga tcagagtatg 2040 tcacagccat cgctaacctg aaacagtgct
cagtctcccc gcttctggaa gcctgtgcct 2100 tcatgatgag gtaaaaccgg
aaaagaagct gcccgcctcc ccaggggcct cagctttccc 2160 tcctcccgtc
ttgattccca gctgccctgg gcctgcctct ctcccttcct gagggcagac 2220
tttgttcagc tcatccgttt tcacaattcc ctcgtgccg 2259 6 703 PRT Sus
scrofa 6 Met Lys Leu Phe Ile Pro Ala Leu Leu Phe Leu Gly Thr Leu
Gly Leu 1 5 10 15 Cys Leu Ala Ala Pro Lys Lys Gly Val Arg Trp Cys
Val Ile Ser Thr 20 25 30 Ala Glu Tyr Ser Lys Cys Arg Gln Trp Gln
Ser Lys Ile Arg Arg Thr 35 40 45 Asn Pro Met Phe Cys Ile Arg Arg
Ala Ser Pro Thr Asp Cys Ile Arg 50 55 60 Ala Ile Ala Ala Lys Arg
Ala Asp Ala Val Thr Leu Asp Gly Gly Leu 65 70 75 80 Val Phe Glu Ala
Asp Gln Tyr Lys Leu Arg Pro Val Ala Ala Glu Ile 85 90 95 Tyr Gly
Thr Glu Glu Asn Pro Gln Thr Tyr Tyr Tyr Ala Val Ala Val 100 105 110
Val Lys Lys Gly Phe Asn Phe Gln Asn Gln Leu Gln Gly Arg Lys Ser 115
120 125 Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile Pro Ile
Gly 130 135 140 Leu Leu Arg Arg Phe Leu Asp Trp Ala Gly Pro Pro Glu
Pro Leu Gln 145 150 155 160 Lys Ala Val Ala Lys Phe Phe Ser Gln Ser
Cys Val Pro Cys Ala Asp 165 170 175 Gly Asn Ala Tyr Pro Asn Leu Cys
Gln Leu Cys Ile Gly Lys Gly Lys 180 185 190 Asp Lys Cys Ala Cys Ser
Ser Gln Glu Pro Tyr Phe Gly Tyr Ser Gly 195 200 205 Ala Phe Asn Cys
Leu His Lys Gly Ile Gly Asp Val Ala Phe Val Lys 210 215 220 Glu Ser
Thr Val Phe Glu Asn Leu Pro Gln Lys Ala Asp Arg Asp Lys 225 230 235
240 Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys Pro Val Glu Ala Phe
245 250 255 Arg Glu Cys His Leu Ala Arg Val Pro Ser His Ala Val Val
Ala Arg 260 265 270 Ser Val Asn Gly Lys Glu Asn Ser Ile Trp Glu Leu
Leu Tyr Gln Ser 275 280 285 Gln Lys Lys Phe Gly Lys Ser Asn Pro Gln
Glu Phe Gln Leu Phe Gly 290 295 300 Ser Pro Gly Gln Gln Lys Asp Leu
Leu Phe Arg Asp Ala Thr Ile Gly 305 310 315 320 Phe Leu Lys Ile Pro
Ser Lys Ile Asp Ser Lys Leu Tyr Leu Gly Leu 325 330 335 Pro Tyr Leu
Thr Ala Ile Gln Gly Leu Arg Glu Thr Ala Ala Glu Val 340 345 350 Glu
Ala Arg Gln Ala Lys Val Val Trp Cys Ala Val Gly Pro Glu Glu 355 360
365 Leu Arg Lys Cys Arg Gln Trp Ser Ser Gln Ser Ser Gln Asn Leu Asn
370 375 380 Cys Ser Leu Ala Ser Thr Thr Glu Asp Cys Ile Val Gln Val
Leu Lys 385 390 395 400 Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly
Phe Ile Tyr Thr Ala 405 410 415 Gly Lys Cys Gly Leu Val Pro Val Leu
Ala Glu Asn Gln Lys Ser Arg 420 425 430 Gln Ser Ser Ser Ser Asp Cys
Val His Arg Pro Thr Gln Gly Tyr Phe 435 440 445 Ala Val Ala Val Val
Arg Lys Ala Asn Gly Gly Ile Thr Trp Asn Ser 450 455 460 Val Arg Gly
Thr Lys Ser Cys His Thr Ala Val Asp Arg Thr Ala Gly 465 470 475 480
Trp Asn Ile Pro Met Gly Leu Leu Val Asn Gln Thr Gly Ser Cys Lys 485
490 495 Phe Asp Glu Phe Phe Ser Gln Ser Cys Ala Pro Gly Ser Gln Pro
Gly 500 505 510 Ser Asn Leu Cys Ala Leu Cys Val Gly Asn Asp Gln Gly
Val Asp Lys 515 520 525 Cys Val Pro Asn Ser Asn Glu Arg Tyr Tyr Gly
Tyr Thr Gly Ala Phe 530 535 540 Arg Cys Leu Ala Glu Asn Ala Gly Asp
Val Ala Phe Val Lys Asp Val 545 550 555 560 Thr Val Leu Asp Asn Thr
Asn Gly Gln Asn Thr Glu Glu Trp Ala Arg 565 570 575 Glu Leu Arg Ser
Asp Asp Phe Glu Leu Leu Cys Leu Asp Gly Thr Arg 580 585 590 Lys Pro
Val Thr Glu Ala Gln Asn Cys His Leu Ala Val Ala Pro Ser 595 600 605
His Ala Val Val Ser Arg Lys Glu Lys Ala Ala Gln Val Glu Gln Val 610
615 620 Leu Leu Thr Glu Gln Ala Gln Phe Gly Arg Tyr Gly Lys Asp Cys
Pro 625 630 635 640 Asp Lys Phe Cys Leu Phe Arg Ser Glu Thr Lys Asn
Leu Leu Phe Asn 645 650 655 Asp Asn Thr Glu Val Leu Ala Gln Leu Gln
Gly Lys Thr Thr Tyr Glu 660 665 670 Lys Tyr Leu Gly Ser Glu Tyr Val
Thr Ala Ile Ala Asn Leu Lys Gln 675 680 685 Cys Ser Val Ser Pro Leu
Leu Glu Ala Cys Ala Phe Met Met Arg 690 695 700 7 36 DNA Homo
sapiens 7 ctgggtcgac gtaggagaag gagtgttcag tggtgc 36 8 24 DNA Homo
sapiens 8 gccgtagact tccgccgcta cagg 24 9 45 DNA Aspergillus oryzae
9 gaggtaccga attcatggtg ttttgatcat tttaaatttt tatat 45 10 45 DNA
Aspergillus oryzae 10 agcagctgca gccaaagcag gtgccgcgac ctgaaggccg
tacag 45 11 27 DNA Aspergillus oryzae 11 ctgcccgggc gtaggagaag
gagtgtt 27 12 34 DNA Homo sapiens 12 catggatcct gttttacgca
atggcctgga taca 34 13 26 DNA Aspergillus oryzae 13 cggaattcat
ggtgttttga tcattt 26 14 52 DNA Aspergillus oryzae 14 tggaattcga
tcgcggatcc gcaatgcatg cagccaaagc aggtgccgcg ac 52
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