U.S. patent application number 10/104793 was filed with the patent office on 2005-09-08 for amino acid modified polypeptides.
Invention is credited to Buechter, Douglas D., Connolly, Kevin, Gruskin, Elliott A., Zhang, Guanghui.
Application Number | 20050196830 10/104793 |
Document ID | / |
Family ID | 22617093 |
Filed Date | 2005-09-08 |
United States Patent
Application |
20050196830 |
Kind Code |
A1 |
Gruskin, Elliott A. ; et
al. |
September 8, 2005 |
Amino acid modified polypeptides
Abstract
Methods are disclosed for simplified recombinant production of
fibrillar collagens. DNAs encoding fibrillar collagen monomers
lacking the N propeptide, the C propeptide, or both propeptides are
introduced into recombinant host cells and expressed. Trimeric
collagen is recovered from the recombinant host cells.
Inventors: |
Gruskin, Elliott A.;
(Killingworth, CT) ; Buechter, Douglas D.;
(Wallingford, CT) ; Zhang, Guanghui; (Guilford,
CT) ; Connolly, Kevin; (Los Angeles, CA) |
Correspondence
Address: |
Mark Farber
Tyco Healthcare Group LP
150 Glover Avenue
Norwalk
CT
06856
US
|
Family ID: |
22617093 |
Appl. No.: |
10/104793 |
Filed: |
March 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10104793 |
Mar 22, 2002 |
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09169768 |
Oct 9, 1998 |
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6492508 |
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09169768 |
Oct 9, 1998 |
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08655086 |
Jun 3, 1996 |
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5821089 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 530/356 |
Current CPC
Class: |
C07K 14/51 20130101;
C07K 14/78 20130101; C12P 21/02 20130101; C07K 14/4725 20130101;
C07K 14/495 20130101; C07K 14/75 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
435/069.1 ;
435/252.3; 530/356 |
International
Class: |
C07K 014/78; C12P
021/02; C12N 001/21 |
Claims
1. Purified human collagen or fragment thereof produced by a
prokaryotic cell, the purified human collagen or fragment thereof
being capable of providing a self aggregate, wherein the purified
human collagen or fragment thereof has incorporated therein at
least one amino acid which has not undergone post translational
enzymatic modification selected from the group consisting of
trans-4-hydroxyproline and 3-hydroxyproline, and the purified human
collagen or fragment thereof having the at least one amino acid
self aggregates to form an extracellular matrix.
2. Human collagen or fragment thereof produced by a prokaryotic
cell according to claim 1 wherein the human collagen or fragment
thereof is encoded for by nucleic acid having the sequence shown in
SEQ. ID. NO. 19.
3. Human collagen or fragment thereof produced by a prokaryotic
cell according to claim 1 wherein the human collagen or fragment
thereof is encoded for by nucleic acid having the sequence shown in
SEQ. ID. NO. 39.
4. Human collagen or fragment thereof produced by a prokaryotic
cell according to claim 1 wherein the human collagen or fragment
thereof is encoded for by nucleic acid having the sequence shown in
SEQ. ID. NO. 43.
5. Human collagen or fragment thereof produced by a prokaryotic
cell according to claim 1 wherein the human collagen or fragment
thereof is encoded for by nucleic acid having the sequence shown in
SEQ. ID. NO. 45.
6. Purified human collagen or fragment thereof produced by a
prokaryotic cell according to claim 1 wherein the purified collagen
or fragment thereof is encoded for by nucleic acid having the
sequence shown in SEQ. ID. NO. 31.
7. Purified human collagen or fragment thereof produced by a
prokaryotic cell, the purified human collagen or fragment thereof
being capable of providing a self aggregate, wherein the purified
human collagen or fragment thereof has incorporated therein at
least one amino acid comprising 3-hydroxyproline which has not
undergone post translational enzymatic modification, and the
purified human collagen or fragment thereof having the at least one
amino acid self aggregates to form an extracellular matrix.
8. Human collagen or a fragment thereof produced by a prokaryotic
cell wherein all naturally occurring sites which contain proline or
hydroxyproline are occupied by trans-4-hydroxyproline or
3-hydroxyproline, wherein the collagen or fragment thereof have the
property of self-aggregation.
9. Human collagen or a fragment thereof according to claim 8,
wherein the human collagen is human type I (.alpha..sub.2)
collagen.
10. Human collagen or a fragment thereof according to claim 9,
wherein the collagen or fragment thereof is encoded by nucleic acid
having the sequence shown in SEQ.ID.NO. 31.
Description
TECHNICAL FIELD
[0001] The invention relates generally to the field of recombinant
protein production, and particularly to the production of
telopeptide collagen in recombinant host cells.
BACKGROUND ART
[0002] Collagen is the major protein component of bone, cartilage,
skin and connective tissue in animals. Collagen in its native form
is typically a rigid, rod-shaped molecule approximately 300 nm long
and 1.5 nm in diameter. It is composed of three collagen
polypeptide monomers which form a triple helix. Mature collagen
monomers are characterized by a long midsection having the
repeating sequence--Gly-X-Y, where X and Y are often proline or
hydroxyproline, bounded at each end by the "telopeptide" regions,
which constitute less than about 5% of the molecule. The
telopeptide regions of the chains are typically responsible for the
crosslinking between the chains (i.e., the formation of collagen
fibrils), and for the immunogenicity of the protein. Collagen
occurs naturally in a number of "types", each having different
physical properties. The most abundant types in mammals and birds
are types I, II and III.
[0003] Mature collagen is formed by the association of three
procollagen monomers which include "pro" domains at the amino and
carboxy terminal ends of the polypeptides. The pro domains are
cleaved from the assembled procollagen trimer to create mature, or
"telopeptide" collagen. The telopeptide domains may be removed by
chemical or enzymatic means to create "atelopeptide" collagen.
[0004] Interestingly, although there are a large number of
different genes encoding for different procollagen monomers, only
particular combinations are produced naturally. For example, skin
fibroblasts synthesize 10 different procollagen monomers
(pro.alpha.1(I), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(I),
pro.alpha.2(V), pro.alpha.3(V), pro.alpha.1(VI), pro.alpha.2(VI),
pro.alpha.3(VI) and pro.alpha.1(VII)), but only 5 types of mature
collagen are produced (types I, III, V, VI and VII).
[0005] Collagen has been utilized extensively in biological
research as a substrate for in vitro cell culture. It has also been
widely used as a component of biocompatible materials for use in
prosthetic implants, sustained drug release matrices, artificial
skin, and wound dressing and wound healing matrices.
[0006] Historically, collagen has been isolated from natural
sources, such as bovine hide, cartilage or bones, and rat tails.
Bones are usually dried, defatted, crushed, and demineralized to
extract collagen, while cartilage and hide are typically minced and
digested with proteolytic enzymes other than collagenase. As
collagen is resistant to most proteolytic enzymes (except
collagenase), this procedure can conveniently remove most of the
contaminating protein that would otherwise be extracted along with
the collagen. However, for medical use, species-matched collagen
(e.g., human collagen for use in human subjects) is highly
desirable in order to minimize the potential for immune response to
the collagen material.
[0007] Human collagen may be purified from human sources such human
placenta (see, for example, U.S. Pat. Nos. 5,002,071 and
5,428,022). Of course, the source material for human collagen is
limited in supply and carries with it the risk of contamination by
pathogens such as hepatitis virus and human immunodeficiency virus
(HIV). Additionally, the material recovered from placenta is biased
as to type and not entirely homogenous.
[0008] Collagen may also be produced by recombinant methods. For
example, International Patent Application No. WO 97/14431 discloses
methods for recombinant production of procollagen in yeast cells
and U.S. Pat. No. 5,593,859 discloses the expression of procollagen
genes in a variety of cell types. In general, the recombinant
production of collagen requires a cloned DNA sequence encoding the
appropriate procollagen monomer(s). The procollagen gene(s) is
cloned into a vector containing the appropriate DNA sequences and
signals for expression of the gene and the construct is introduced
into the host cells. Optionally, genes expressing a
prolyl-4-hydroxylase alpha subunit and a protein disulfide
isomerase are also introduced into the host cells (these are the
two subunits which make up prolyl-4-hydroxylase). Addition of the
prolyl-4-hydroxylase leads to the conversion of some of the prolyl
residues in the procollagen chains to hydroxyproline, which are
important in interchain bonds of the triple helix and increase the
thermal stability of the protein.
[0009] Alternately, recombinant collagen may be produced using
transgenic technology. Constructs containing the desired collagen
gene linked to the appropriate promoter/enhancer elements and
processing signals are introduced into embryo cells by the
formation of ES cell chimera, direct injection into oocytes, or any
other appropriate technique. Transgenic production of recombinant
collagen is particularly advantageous when the collagen is
expressed in milk (i.e., by mammary cells), such as described in
U.S. Pat. No. 5,667,839 to Berg. However, the production of
transgenic animals for commercial production of collagen is a long
and expensive process.
[0010] One difficulty of recombinant expression of collagen is the
processing of the "pro" regions of procollagen monomers. It is
widely accepted that folding of the three monomers to form the
trimer begins in the carboxylpro-region ("C propeptide") and that
the C propeptide contains signals responsible for monomer selection
(Bachinger et al., 1980, Eur. J. Biochem., 106:619-632; Bachinger
et al., 1981, J. Biol. Chem. 256:13193-13199). One group has
identified a region in the carboxy pro-region that they believe is
necessary and sufficient for monomer selection (Bulleid et al.,
1997, EMBO J. 16(22):6694-6701; Lees et al., 1997, EMBO J.
16(5):908-916; International Patent Application No. WO 97/08311;
McLaughling et al., 1998, Matrix Biol. 16:369-377). Additionally,
Lee et al. (1992, J Biol. Chem. 267(33):24126-24133) have shown
that deletion of the N propeptide results in decreased secretion of
human .alpha.1 pC collagen from CHL cells, but not Mov-13 cells.
Accordingly, it is believed that the pro-regions must be retained
for proper chain selection, alignment and folding of collagen
produced by recombinant methods. In cells which normally produce
collagens, specific proteolytic processing enzymes are produced
which remove the N and C propeptides following the secretion of
collagen. These enzymes are not present in cells which do not
normally produce collagen (including commonly used recombinant host
cells such as bacteria and yeast).
[0011] Ideally, the recombinant production of collagen is
accomplished with a recombinant host cell system that has a high
capacity and a relatively low cost (such as bacteria or yeast).
Because bacteria and yeast do not normally produce the enzyme
necessary for processing of the N and C propeptides, the
propeptides must be removed after recovering the recombinant
procollagen from the host cells. This can be accomplished by the
use of pepsin, but processing with pepsin produces "ragged" ends
that do not correspond to the ends of mature collagen secreted by
mammalian cells which normally produce fibrillar collagen.
Alternately, the enzymes which process the N and C propeptides can
be produced and used to remove the propeptides. Any contamination
of these preparations with other proteases will result in ragged
ends. This added processing step increases the cost and decreases
the convenience of production in these otherwise desirable host
cell systems. Accordingly, there is a need in the art for
simplified methods of producing genuine telopeptide collagen in
high capacity systems.
DISCLOSURE OF THE INVENTION
[0012] The inventors have discovered new methods for the
recombinant production of fibrillar collagens. The inventors have
surprisingly and unexpectedly found that co-expression of DNA
constructs encoding .alpha.1(I) and .alpha.2(I) collagen monomers
lacking the N and C propeptides form heterotrimeric telopeptide
collagen having the properties of genuine human type I collagen.
Additionally, the inventors have found that co-expression in yeast
of DNA constructs encoding a non-collagen signal sequence linked to
.alpha.1(I) and .alpha.2(I) collagen monomers lacking the N
propeptide results in a surprising increase in the production of
"pC collagen" (procollagen trimer lacking the N propeptide).
Additionally, co-expression in yeast of DNA constructs encoding a
non-collagen signal sequence linked to .alpha.1(I) and .alpha.2(I)
collagen monomers lacking the N and C propeptides results in a
surprising increase in the production of type I collagen.
[0013] The methods of the instant invention may be used to produce
any of the fibrillar collagens (e.g., types 1-III, V and XI) from
any species, but are particularly useful for the production of
recombinant human collagens for use in medical applications.
[0014] In one embodiment, the invention relates to methods for
producing fibrillar collagen by culturing a recombinant host cell
comprising a DNA encoding a fibrillar collagen monomer lacking a C
propeptide sequence selection and alignment domain (SSAD) under
conditions appropriate for expression of said DNA; and producing
fibrillar collagen. The DNA may encode any of the fibrillar
collagen monomers, such as .alpha.1(I), .alpha.2(I), .alpha.1(II),
.alpha.1(III), .alpha.1(V), .alpha.2(V), .alpha.3(V), .alpha.1(XI),
.alpha.2(XI), and .alpha.3(XI). Optionally, the DNA encoding the
fibrillar collagen monomer lacking a C propeptide SSAD may also
lack DNA encoding the N propeptide.
[0015] In another embodiment, the invention relates to methods for
producing fibrillar collagen by culturing a recombinant yeast host
cell comprising a DNA encoding a fibrillar collagen monomer lacking
a N propeptide under conditions appropriate for expression of said
DNA; and producing fibrillar collagen.
[0016] Another embodiment relates to recombinant host cells
comprising an expression construct comprising a DNA encoding a
fibrillar collagen monomer lacking a C propeptide sequence
selection and alignment domain (SSAD). The DNA may encode any of
the fibrillar collagen monomers, such as .alpha.1(I), .alpha.2(I),
.alpha.1(II), .alpha.1(III), .alpha.1(V), .alpha.2(V), .alpha.3(V),
.alpha.1(XI), .alpha.2(XI), and .alpha.3(XI). Optionally, the DNA
encoding the fibrillar collagen monomer lacking a C propeptide SSAD
may also lack DNA encoding the N propeptide.
[0017] In a further embodiment, the invention relates to trimeric
collagen molecules which lack propeptide domains and lack any
glycosylation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows an alignment of SSAD sequences, shown in single
letter amino acid code, as identified by Lees et al. (1997, supra).
Positions 1-12 and 21-23 are considered the essential positions in
the SSAD.
[0019] FIG. 2 shows a map of shuttle vector plasmid Gp5432.
[0020] FIG. 3 shows the amino acid sequence of human
prepro.alpha.1(I) collagen posted to Genbank under accession number
AF017178. The signal sequence (pre domain) is underlined. The first
amino acid of the N telopeptide is marked with an "*". The last
amino acid of the C telopeptide is marked with a "#".
[0021] FIG. 4 shows the amino acid sequence of human
prepro.alpha.2(I) collagen posted to Genbank under accession number
Z74616. The signal sequence (pre domain) is underlined. The first
amino acid of the N telopeptide is marked with an "*". The last
amino acid of the C telopeptide is marked with a "#".
[0022] FIG. 5 shows a half-tone reproduction of a western blot
demonstrating results from a thermal stability protease assay.
Lanes labeled HSF are samples of type I collagen from medium
conditioned by human skin fibroblasts. Lanes labeled CYT29 are
collagen produced in yeast using an expression construct encoding
preproHSA.alpha.1(I) and preproHSA.alpha.2(I) (preproHSA.alpha.1(I)
and preproHSA.alpha.2(I) comprise the human serum albumin signal
sequence plus four amino acids of the pro domain linked to a KEX2
cleavage site fused to the .alpha.1(I) and .alpha.2(I) telopeptide
collagen monomers).
[0023] FIG. 6 shows a half-tone reproduction of a western blot
demonstrating results from a mammalian collagenase digest of human
skin fibroblast and yeast-derived collagen. Lanes labeled HSF are
samples of type I collagen from human skin fibroblasts. Lanes
labeled CYT29 are collagen produced in yeast using an expression
construct encoding preproHSA.alpha.1(I) and
preproHSA.alpha.2(I).
[0024] FIG. 7 shows a map of shuttle vector plasmid Gp5102.
BEST MODE FOR CARRYING OUT THE INVENTION
[0025] The methods of the instant invention generally involve the
use of recombinant host cells comprising DNA expression constructs
encoding the production of fibrillar collagen monomers lacking at
least portions of one or both of the propeptides. The recombinant
host cells are incubated under conditions appropriate for the
expression of the constructs, and trimeric telopeptide collagen is
recovered.
[0026] Definitions
[0027] As used herein, the term "collagen" refers to a family of
homotrimeric and heterotrimeric proteins comprised of collagen
monomers. There are a multitude of known collagens (at least 19
types) which serve a variety of functions in the body. There are an
even greater number of collagen monomers, each encoded by a
separate gene, that are necessary to make the different collagens.
The most common collagens are types I, II, and III. Collagen
molecules contain large areas of helical structure, wherein the
three collagen monomers form a triple helix. The regions of the
collagen monomers in the helical areas of the collagen molecule
generally have the sequence GXY, where G is glycine and X and Y are
any amino acid, although most commonly X and Y are proline and/or
hydroxyproline. Hydroxyproline is formed from proline by the action
of prolyl-4-hydroxylase.
[0028] As used herein, the term "fibrillar collagen" means a
collagen which can normally form collagen fibrils. The fibrillar
collagens are collagen types I-III, V, and XI. The collagen
monomers that make up the fibrillar collagens contain "telopeptide"
regions at the amino (N) and carboxy (C) terminal ends of the
monomers which are non-helical in the collagen trimer. These
collagens self-assemble into fibrils with the C-terminal end of the
helical domain and the C propeptide of one collagen triple helix
overlapping with the N telopeptide and the N-terminal end of the
triple helical domain of an adjacent collagen molecule. The
monomers that make up the fibrillar collagens are made as
preproproteins, including an N-terminal secretion signal sequence
and N and C-terminal propeptide domains. The signal sequence is
normally cleaved by signal peptidase, as with most secreted
proteins, and the propeptides are removed by specific proteolytic
processing enzymes after association, folding and secretion of
trimeric collagen. The term fibrillar collagen encompasses both
native (i.e., naturally occurring) and variant fibrillar collagens
(i.e., fibrillar collagens with one or more alterations in the
sequence of one or more of the fibrillar collagen monomers).
[0029] The term "sequence selection and alignment domain" or "SSAD"
refers to a portion of the C propeptide of fibrillar collagens
identified by Lees et al. (1997, supra) as responsible for chain
selection and alignment. SSAD sequences for .alpha.1(I),
.alpha.2(I), .alpha.1(II), .alpha.1(III), .alpha.1(V), .alpha.2(V),
.alpha.1(XI), and .alpha.2(XI) have been identified in Lees et al.
and are shown in FIG. 1. Only positions 1-12 and 21-23 of the
sequences shown in FIG. 1 are considered part of the SSAD. SSADs
from other fibrillar collagen monomers can easily be identified in
the C propeptide of fibrillar collagen monomers by sequence
similarity alignment with the SSADs shown in FIG. 1.
[0030] The term "DNA encoding a fibrillar collagen monomer", as
used herein, means a DNA sequence which encodes a collagen monomer
that is a component of a fibrillar collagen and which lacks the N
propeptide domain, the SSAD, or both. cDNAs encoding fibrillar
collagen monomers have been identified, cloned and sequenced, and
are readily available to the research community through Genbank and
other DNA sequence depositories. Due to the large size of the
collagen monomers, the primary source of sequence information is
cloned DNA sequence. By conceptual translation, the amino acid
sequence of the fibrillar collagen monomers can be deduced. A DNA
encoding a fibrillar collagen monomer is any DNA sequence that
encodes the amino acid sequence of a fibrillar collagen monomer.
Due to the degeneracy of the DNA code, a large number of different
DNA sequences will be useful for the expression of any given
fibrillar collagen monomer. Additionally, due to codon usage bias,
the DNAs useful in the instant invention may be selected to be
particularly advantageous for use in particular host cell (e.g.,
for use in S. cerevisiae, DNAs encoding fibrillar collagen monomers
may be selected or synthesized which utilize codons that are
preferred in S. cerevisiae).
[0031] DNA encoding any collagen monomer that is a component of
fibrillar collagen may be useful in the methods of the instant
invention. Particularly preferred collagen monomers are
.alpha.1(I), .alpha.2(I), .alpha.1(II), .alpha.1(II), .alpha.1(V),
.alpha.2(V), .alpha.3(V), .alpha.1(XI), .alpha.2(XI), and
.alpha.3(XI), more preferably the human forms of .alpha.1(I),
.alpha.2(I), .alpha.1(II), .alpha.1(III), .alpha.1(V), .alpha.2(V),
.alpha.3(V), .alpha.1(XI), .alpha.2(XI), and .alpha.3(XI). The
amino acid sequences for these proteins are available to the public
(see, for example, Tromp et al., 1988, Biochem J. 253(3):919-922;
Kuivaniemi et al., 1988, Biochem J. 252(3):633-640; Su et al.,
1989, Nucleic Acid Res. 17(22):9473; Ala-Kokko et al., 1989,
Biochem. J. 260(2):509-516; Takahara et al., 1991, J. Biol. Chem.
266(20): 13124-13129; Weil et al., 1987, Nucleic Acid Res.
15(1):181-198; Bernard et al., 1988, J. Biol. Chem.
263(32):17159-17166; Kimuraet al., 1989, J. Biol. Chem.
264(23):13910-13916; Mann et al., 1992, Biol. Chem. Hoppe Seyler
373:69-75; Sandell et al., 1991, J. Cell. Biol., 114:1307-1319).
Additionally, deletion mutants of fibrillar collagens such as that
described in Sieron et al. (1993, J. Biol. Chem.
268(28):21232-21237) and D period deletions such as described in
Zafarullah et al. (1997, Matrix Biol. 16:245-253) and Arnold et al.
(1997, Matrix Biol. 16:105-116) may also be produced by the method
of the instant invention. The DNAs may be obtained by any method
from any source known in the art, such as isolation from cDNA or
genomic libraries, chemical synthesis, or amplification from any
available template. Additionally, DNAs encoding variants may be
produced by de novo synthesis or by modification of an existing DNA
by any of the methods known in the art.
[0032] DNA encoding fibrillar collagen monomers for use in
accordance with the instant invention lack sequences encoding the N
propeptide, the C propeptide SSAD, or both. Lees et al. (1997,
supra) teach that the SSAD domain is required for proper chain
selection and association of collagen monomers. Preferably, DNAs
encoding fibrillar collagen monomers lack the SSAD and also lack
sequence encoding at least 50% of the total C propeptide domain,
more preferably at least 75% of the total C propeptide domain, and
even more preferably total 90% of the propeptide domain, and most
preferably DNAs encoding fibrillar collagen monomers lack all of
the C propeptide domain. Alternately, the DNA encoding fibrillar
collagen monomers may lack sequence encoding part or all of the N
propeptide domain. Preferred deletions of the sequence encoding the
N propeptide domain include DNAs lacking sequence encoding 50%,
75%, 90% or all of the N propeptide. Additionally, DNA encoding
fibrillar collagen monomers may lack sequence encoding portions of
or the entirety of the N and C propeptides. Preferably, the DNA
encoding fibrillar collagens for use in accordance with the instant
invention lack sequences encoding both the N and C propeptides. The
boundaries of the mature peptide and the N and C propeptides are
well known in the art.
[0033] For use in the instant invention, the DNA encoding a
fibrillar collagen monomer is cloned into an expression construct.
General techniques for nucleic acid manipulation useful for the
practice of the claimed invention are described generally, for
example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, Vols. 1-3 (Cold Spring Harbor Laboratory Press, 2 ed.,
(1989); or F. Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
(Green Publishing and Wiley-Interscience: New York, 1987) and
periodic updates.
[0034] The exact details of the expression construct will vary
according to the particular host cell that is to be used as well as
to the desired characteristics of the expression system, as is well
known in the art. For example, for production in S. cerevisiae, the
DNA encoding a fibrillar collagen monomer is placed into operable
linkage with a promoter that is operable in S. cerevisiae and which
has the desired characteristics (e.g., inducible/derepressible or
constituitive). Where bacterial host cells are utilized, promoters
and promoter/operators such as the araB, trp, lac, gal, tac (a
hybrid of the tip and lac promoter/operator), T7, and the like are
useful in accordance with the instant invention. Acceptable
promoters for use in the instant invention where the host cell is
S. cerevisiae include, but are not limited to GAL1-10, PHO5, PGK1,
GDP1, chelatin, PMA1, MET3, CUP1, GAP, TPI, MF.alpha.1 and
MF.alpha.2, as well as the hybrid promoters PGK/.alpha.2,
TPI/.alpha.2, GAP/GAL, PGK/GAL, GAP/ADH2, GAP/PHO5, ADH2PHO5,
CYC1/GRE, and PGK/ARE, and other promoters active in S. cerevisiae
as are known in the art. Where S. pombe is utilized as the host
cell, promoters such as FBP1, NMT1, ADH1 and other promoters active
in S. pombe as are known in the art, such as the human
cytomegalovirus (hCMV) LTR. The AOX1 promoter is preferred when
Pichia pastoris is the host cell, although other promoters known in
the art, such as GAP and PGK are also acceptable. Further guidance
with regard to features of expression constructs for yeast host
cells may be found in, for example, Romanos et al. (1992, Yeast
8:423-488). When other eukaryotic cells are the desired host cell,
any promoter active in the host cell may be utilized. For example,
when the desired host cell is a mammalian cell line, the promoter
may be a viral promoter/enhance (e.g., the herpes virus thymidine
kinase (TK) promoter or a long terminal repeat (LTR), such as the
LTR from cytomegalovirus (CMV), Rous sarcoma virus (RSV) or mouse
mammary tumor virus (MMTV)) or a mammalian promoter, preferably an
inducible promoter such as the metallothionein or glucocorticoid
receptor promoters and the like.
[0035] Expression constructs may also include other DNA sequences
appropriate for the intended host cell. For example, expression
constructs for use in higher eukaryotic cell lines (e.g.,
vertebrate and insect cell lines) will include a poly-adenylation
site and may include an intron (including signals for processing
the intron), as the presence of an intron appears to increase mRNA
export from the nucleus in many systems. Additionally, a secretion
signal sequence operable in the host cell is normally included as
part of the construct. The secretion signal sequence may be from a
collagen monomer gene or from a non-collagen gene. In one preferred
embodiment, the secretion signal sequence is a prepro sequence
derived from human serum albumin which additionally contains a KEX2
protease processing site (MKWVTFISLLFLFSSAYSRGVFRR in single letter
amino acid code, the signal peptidase site is between S and R, RGVF
is derived from the HSA pro domain). If the secretion signal
sequence is derived from a collagen monomer gene, it may be form a
fibrillar collagen monomer (and may be derived from the same
protein as the DNA encoding the fibrillar collagen monomer to be
expressed or from a different fibrillar collagen monomer) or a
non-fibrillar collagen monomer. Where the expression construct is
intended for use in a prokaryotic cell, the expression construct
should include a signal sequence which directs transport of the
synthesized peptide into the periplasmic space.
[0036] Preferably, the expression construct will also comprise a
means for selecting for host cells which contain the expression
construct (a "selectable marker"). Selectable markers are well
known in the art. For example, the selectable marker may be a
resistance gene, such as a antibiotic resistance gene (e.g., the
neo gene which confers resistance to the antibiotic gentamycin), or
it may be a gene which complements an auxotrophy of the host cell.
If the host cell is a yeast cell, the selectable marker is
preferably a gene which complements an auxotrophy of the cell (for
example, complementing genes useful in S. cerevisiae, P. pastoris
and S. pombe include LEU2, TRP1, TRP1d, URA3, URA3d, HIS3, HIS4,
ARG4, LEU2d), although antibiotic resistance markers such as SH
BLE, which confers resistance to zeocin, may also be used. If the
host cell is a prokaryotic or higher eukaryotic cell, the
selectable marker is preferably an antibiotic resistance marker
(e.g., neo.sup.r or bla). Alternately, a separate selectable marker
gene is not included in the expression vector, and the DNA encoding
the fibrillar collagen monomer is used as a selectable marker (upon
induction or derepression for controllable promoters, or after
transfection for a constituitive promoter, fluorescence-activated
cell sorting, FACS, may be used to select those cells which express
the recombinant collagen). Preferably, the expression construct
comprises a separate selectable marker gene.
[0037] The expression construct may also contain sequences which
act as an "ARS" (autonomous replicating sequence) which will allow
the expression construct to replicate in the host cell without
being integrated into the host cell chromosome. Origins of
replication for bacterial plasmids are well known. ARS for use in
yeast cells are also well known (the 2.mu. origin of replication
and operative fragments thereof, especially the full length
sequence 2.mu. is preferred, see, for example International Patent
Application No. WO 97/14431, although CEN-based plasmids and YACS
are also useful in the instant invention) and ARS which act in
higher mammalian cells have been recently described (see, for
example, Pelletier et al., 1997, J. Cell. Biochem. 66(1):87-97)).
Alternately, the expression construct may include DNA sequences
which will direct or allow the integration of the construct into
the host cell chromosome by homologous or site-directed
recombination.
[0038] Where the host cell is a eukaryotic cell, it may be
advantageous for the expression vector to be a "shuttle vector",
because manipulation of DNA is substantially more convenient in
bacterial cells. A shuttle vector is one which carries the
necessary signals to for manipulations in bacteria as well as the
desired host cell. So, for example, the expression construct may
also comprise an ARS ("ori") which acts in prokaryotic cells as
well as a selectable marker which is useful for selection of
prokaryotic cells.
[0039] The host cells for use in the instant invention may be any
convenient host cell, including bacterial, yeast, and eukaryotic
cells. Yeast and higher eukaryotic cells are preferred host cells.
For yeast host cells, Saccharomyces cerevisiae, Pichia pastoris,
Hansenula polymorpha, Kluyveromyces lactis, Schwanniomyces
occidentis and Yarrowia lipolytica strains are preferred. Of the
higher eukaryotic cells, insect cells such as sf9 are preferred, as
are mammalian cell lines which produce non-fibrillar collagens and
do not produce any endogenous fibrillar collagens, such as HT-1080,
293, and NSO cells.
[0040] If the host cell does not have prolyl-4-hydroxylase activity
(or has insufficient activity as is the case in insect cells), the
host cell preferably is altered to produce prolyl-4-hydroxylase.
This may be conveniently accomplished by introducing expression
constructs coding for the expression of the subunits of
prolyl-4-hydroxylase into the host cell. Prolyl-4-hydroxylase is a
tetramer comprising two alpha subunits and two beta subunits
(.alpha..sub.2.beta..sub.2). The beta subunit is also known as
protein disulfide isomerase (PDI). Expression constructs for
prolyl-4-hydroxylase have been described for yeast (Vuorela et al.,
1997, EMBO J. 16(22):6702-6712) and for insect cells (Lamberg et
al., 1996, J. Biol. Chem. 271(20):11988-11995). In the case of a
bacterial host cell, the expression construct for
prolyl-4-hydroxylase will preferably incorporate a translocation
signal to direct the transport of the subunits of the enzyme to the
periplasmic space. Alternately, the prolyl-4-hydroxylase expression
construct may be included in the fibrillar collagen monomer
construct. In this arrangement, the expression construct may direct
the production of separate messages for the fibrillar collagen
monomer and the prolyl-4-hydroxylase subunits or it may direct the
production of a polycistronic message. Separate messages are
preferred for eukaryotic hosts, while the expression of a
polycistronic message is preferred for prokaryotic hosts.
[0041] The expression construct is introduced into the host cells
by any convenient method known to the art. For example, for yeast
host cells, the construct may be introduced by electroporation,
lithium acetate/PEG and other methods known in the art. Higher
eukaryotes may be transformed by electroporation, microprojectile
bombardment, calcium phosphate transfection, lipofection, or any
other method known to the art. Bacterial host cells may be
transfected by electroporation, calcium chloride-mediated
transfection, or any other method known in the art.
[0042] After introduction of the expression construct into the host
cell, host cells comprising the expression construct are normally
selected on the basis of the selectable marker that is included in
the expression vector. As will be apparent, the exact details of
the selection process will depend on the identity of the selectable
marker. If the selectable marker is an antibiotic resistance gene,
the transfected host cell population is generally cultured in the
presence of an antibiotic to which resistance is conferred by the
selectable marker. The antibiotic eliminates those cells which are
not resistant (i.e., those cells which do not carry the resistance
gene) and allows the propagation of those host cells which carry
the resistance gene (and presumably carry the rest of the
expression construct as well). If the selectable marker is a gene
which complements an auxotrophy of the host cells, then the
transfected host cell population is cultured in the absence of the
compound for the host cells are auxotrophic. Those cells which are
able to propagate under these conditions carry the complementing
gene and thus presumably carry the rest of the expression
construct.
[0043] Host cells which pass the selection process may be "cloned"
according to any method known in the art that is appropriate for
the host cell. For microbial host cells such as yeast and bacteria,
the selected cells may be plated on solid media under selection
conditions, and single clones may be selected for further
selection, characterization or use. Higher eukaryotic cells are
generally further cloned by limiting dilution. This process may be
carried out several times to ensure the stability of the expression
construct within the host cell.
[0044] For production of trimeric collagen, the recombinant host
cells comprising the expression construct are generally cultured to
expand cell numbers. This expansion process may be carried out in
any appropriate culturing apparatus known to the art. For yeast and
bacterial cells, an apparatus as simple as a shaken culture flask
may be used, although large scale culture is generally carried out
in a fermenter. For insect cells, the culture is generally carried
out in "spinner flasks" (culture vessels comprising a means for
stirring the cells suspended in a liquid culture medium). For
mammalian cell lines, the cells may be grown in simple culture
plates or flasks, but as for the yeast and bacterial host cells,
large scale culture is generally performed in a specially adapted
apparatus, a variety of which are known in the art.
[0045] If the host cells comprise (either naturally or by
introduction of the appropriate expression constructs)
prolyl-4-hydroxylase, then vitamin C (ascorbic acid or one of its
salts) may be added to the culture medium, although applicants have
found ascorbate may not be necessary if the recombinant host cells
are S. cerevisiae cells. If ascorbic acid is added, it is generally
added to a concentration of between 10-200 .mu.g/ml, preferably
about 80 .mu.g/ml. If ascorbate is to be added, it need not be
added until the host cells begin producing recombinant
collagen.
[0046] The recombinant host cells are cultured under conditions
appropriate for the expression of the DNA encoding the fibrillar
collagen monomer. If the expression construct utilizes a
controllable expression system, the expression of the DNA encoding
the fibrillar collagen monomers is induced or derepressed, as is
appropriate for the particular expression construct. The exact
method of inducing or derepressing the expression of the DNA
encoding the fibrillar collagen monomers will depend on the
properties of the particular expression construct used and the
identity of the host cell, as will be apparent to one of skill in
the art. Generally, for inducible promoters, a molecule which
induces expression is added to the culture medium. For example, in
yeast transfected with an expression vector utilizing the GAL1-10
promoter, galactose is added to the culture medium in the absence
of dextrose. In bacteria utilizing an expression vector with the
lac promoter, isopropyl-.beta.-D-thiogalactopyranoside (IPTG) is
added to the medium to derepress expression. For constituitive
promoters, the cells are cultured in a medium providing the
appropriate environment and sufficient nutrients to support the
survival of the cells and the synthesis of the fibrillar collagen
monomers.
[0047] Mature fibrillar collagen is produced by the recombinant
host cells. Surprisingly, the fibrillar collagen monomers assemble
into mature collagen trimers in the absence of the C
propeptide.
[0048] Fibrillar collagen may then be recovered from the culture.
The exact method of recovery of the collagen from the culture will
depend on the host cell type and the expression construct. In many
microbial host cells, the collagen will be trapped within the cell
wall of the recombinant host cell, even though it has been
transported out of the cytoplasm. In this instance, the host cells
are preferably disrupted to recover the fibrillar collagen.
Disruption may be accomplished by any means known in the art,
including sonication, microfluidization, lysis in a french press or
similar apparatus, disruption by vigorous agitation with glass
beads, and the like. Alternately, in higher eukaryotic cells or
microbial cells having mutations which render them "leaky", the
fibrillar collagen may be recovered by collection of the culture
medium.
[0049] When DNAs encoding collagen monomers lacking the N and C
propeptides are utilized in yeast or prokaryotic cells in
accordance with the methods of the instant invention,
non-glycosylated trimeric collagen having genuine N and C terminal
ends (i.e., the N and C telopeptide ends found in fibrillar
collagens secreted from mammalian cells that normally produce
fibrillar collagen) is produced.
[0050] The patents, patent applications, and publications cited
throughout the disclosure are incorporated herein by reference in
their entirety.
EXAMPLES
Example 1
Recombinant Production of Type I Telopeptide Collagen
[0051] Recombinant type I telopeptide collagen (.alpha.1 homotrimer
and .alpha.1/.alpha.2 heterotrimer) was produced in S. cerevisiae
host cells using expression constructs coding for human .alpha.1(I)
and .alpha.2(I) collagen monomers. A number of different shuttle
vectors were created, most based on Gp5432 (see FIG. 2 for a map of
Gp5432) which contains DNA encoding the preprocollagen .alpha.1(I)
and .alpha.2(I) monomers operably linked to the bidirectional
GAL1-10 promoter (the sequences of prepro.alpha.1(I) and
prepro.alpha.2(I) are shown in FIGS. 3 and 4, respectively). The
PGK terminator (PGKt) is supplied at the 3' end of the .alpha.2(I)
sequence, while a terminator in the 2.mu. ARS (from the FLP gene)
acts to terminate transcription of the .alpha.1(I) gene. Gp5432
also contains a yeast selectable marker (TRP1), an operable 1.6 kb
fragment of the 2.mu. yeast origin, a bacterial ori, and a
bacterial selectable marker (bla). Additionally, a construct was
made based on Gp5102, which is very similar to Gp5432 but does not
contain the .alpha.2(I) sequence or the PGKt (see FIG. 7 for a map
of Gp5102). Constructs were created from Gp5432 which: (a) replaced
the collagen secretion signal sequence (the "pre" domain) with a
prepro domain from human serum albumin (HSA) which additionally
contains a KEX2 protease processing site (MKWVTFISLLFLFSSAYSRGVFRR
in single letter amino acid code (the KEX2 cleaves at the
carboxy-end of RR), designated pGET462); (b) encoded pC .alpha.1(I)
and pC .alpha.2(I) linked to the preproHSA/KEX2 sequence
(designated pDO243880); and (c) and constructs with the .alpha.1(I)
and .alpha.2(I) mature domain (i.e., the signal sequence and the N
and C propeptides were deleted from the preproCOL1A1 and
preproCOL1A2) linked to the preproHSA/KEX2 sequence or their native
signal sequences (designated pDO248053 and pDO248098,
respectively). pDO248010 was created from Gp5102, and encodes the
.alpha.1(I) telopeptide sequence linked the preproHSA/KEX2
sequence.
[0052] The expression constructs were transformed into GY 5361 by
electroporation. This host strain also contained a
chromosomally-integrated expression construct encoding for the two
subunits of chicken prolyl-4-hydroxylase. The alpha subunit (Bassuk
et al., 1989, Proc. Natl. Acad. Sci. USA 86:7382-7386) and beta
subunit, also known as PDI (Kao et al., 1988, Conn. Tiss. Res.
18:157-174), were cloned into an expression construct under the
control of the bidirectional GAL1-10 promoter. The
prolyl-4-hydroxylase construct also included the URA3 selectable
marker and sequences from the TRP1 gene to allow integration by
homologous recombination. Correct integrants were TRP1.DELTA..
[0053] After electroporation of GY5361 with 100 ng of plasmid DNA,
transformants were selected on 2% agar plates containing 2%
dextrose, 0.67% yeast nitrogen base lacking amino acids (YNB), 0.5%
casamino acids by growing 3 days at 30.degree. C. Transformants
were grown overnight at 30.degree. C. in media containing 2%
dextrose, 0.67% YNB, 0.5% casamino acids to an OD.sub.600 of 3
(approximately 1.times.10.sup.8 cells/ml). To induce collagen
expression, the overnight cultures (in glucose-containing media)
were dilute to OD.sub.600 of approximately 0.05 in media containing
0.5% galactose, 0.5% dextrose, 0.67% YNB and 0.5% casamino acids,
1% sodium citrate, pH 6.5, 50 mM sodium ascorbate, 300 mM
.alpha.-ketoglutarate, 100 mM ferric chloride (FeCl3), 100 mM
glycine, 100 mM proline. Inductions were allowed to proceed for
48-96 hours at 30.degree. C.
[0054] Cells were harvested by centrifugation, resuspended in 0.1 M
tris HCl, pH 7.4, 0.4 M NaCl, 10 mM EDTA and lysed by vortexing in
a centrifuge tube with glass beads. The beads and cellular debris
were removed by centrifugation. Production of type I collagen was
measured by immunoassay and protease sensitivity.
[0055] Protein yield was determined using a luminometric
immunoassay. The assay utilizes a goat anti-type I collagen
antibody commercially available from Biodesign International
(Kennebunk, Me.) derivatized with either biotin or ruthenium
chelate. Samples were diluted from 1:40 to 1:60 in "Matrix buffer"
(100 mM PIPES, pH 6.8, and 1% w/v bovine serum albumin) and 25
.mu.l samples were dispensed into tubes. 50 .mu.l of an antibody
working solution containing 1 .mu.g/ml of ruthenium chelate
conjugated antibody and 1.5 .mu.g/ml biotin conjugated antibody in
diluent (Matrix buffer plus 1.5% Tween-20) was added to each tube
and the tubes were incubated for two hours at room temperature
(approximately 20.degree. C.). After the incubation, 25 .mu.l of a
1 mg/ml solution of streptavidin-conjugated magnetic beads (in
diluent) were added to each tube. The tubes were shaken or vortexed
for 30 seconds. 200 .mu.l of assay buffer (ORIGEN assay buffer,
Igen, Inc., catalog number 402-050-01) was added to each tube and
the tubes were mixed then placed in a ORIGEN analyzer (Igen, Inc.,
model #1100-1000). Results are shown below in Table 1.
1TABLE 1 Expression levels (.mu.g collagen/ Strain Proteins encoded
mg protein) CYT 30 preproCOL.alpha.1(I)/preproCOLa2(I) 0.68 .+-.
0.046 CYT 31 preproHSApro.alpha.1(I)/preproHSApro.alpha.2(I) 0.43
.+-. 0.015 CYT 32 preproHSApC.alpha.1(I)/preproHSApro.alpha.2(I)
1.21 .+-. 0.19 CYT 33 preproHSA.alpha.1(I)/preproHSA.alpha.2(I)
1.50 .+-. 0.038 CYT 44 preCOL.alpha.1(I)/preCOL.alpha.2(I) 0.13
.+-. 0.022
[0056] The The constructs expression .alpha.1 and .alpha.2 linked
to their native signal sequences gave reduced expression, which is
believed to be due to an alteration of the amino acid context at
the signal peptidase cleavage site, which impairs signal peptide
processing.
[0057] The collagens were also tested by proteolytic assays for
thermal stability. Resistance to pepsin or trypsin/chymotrypsin was
measured by the method of Bruckner et al. (1981, Anal. Biochem.
110:360-368). Basically, samples were incubated with protease at a
series of temperatures (4, 20, 25, 30 and 35.degree. C. for pepsin
and 20, 25, 30 and 35.degree. C. for trypsin/chymotrypsin). Type I
collagen from human skin fibroblasts was incubated with pepsin or
trypsin/chymotrypsin as a standard. Results were assayed by western
blotting (Towbin et al., 1979, Proc. Natl. Acad. Sci. USA
76:4350-4354) using a rabbit anti-type I collagen antibody from
Rockland, Inc. (Gilbertsville, Pa.), detected with a
peroxidase-labeled goat anti-rabbit IgG (H+L) and visualized with a
chemiluminescent reaction (ECL Western Blotting Kit, Amersham,
Inc.). Assay results for .alpha.1(I)/.alpha.2(I) heterotrimer are
shown in FIG. 5. .alpha.1(I) homotrimer had equivalent thermal
stability as measured by this assay (data not shown).
[0058] In this assay, the triple helical portions of the collagen
trimer are resistant to protease digestion. As the temperature is
increased to the melting point of the triple helical region, the
triple helical portions of the molecule become susceptible to
proteolytic digestion. Monomeric collagen chains and improperly
folded collagen monomers are highly susceptible to protease at low
temperatures. These results show that the collagen produced by
expression of DNA encoding .alpha.1(I) and .alpha.2(I) collagen
lacking the N and C propeptides is approximately equivalent to
human skin fibroblast type I collagen with regards to thermal
stability and protease resistance.
[0059] The correct folding and register of the three monomers in
the yeast-produced triple helical collagen was assayed by digestion
with mammalian collagenase. Human skin fibroblast collagenase
cleaves each of the three chains of collagen at a single point.
Collagenase is highly sensitive to local structure and sequence at
the cleavage site. If the molecule is improperly folded or the
chains are folded out of register, collagenase will not cleave (Wu
et al., 1990, Proc. Natl. Acad. Sci. USA 78:5888-5892). Samples
were digested with purified human fibroblast collagenase in 0.05 M
tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M CaCl.sub.2 for 16 hours at
25.degree. C. Prior to use in the assay, procollagenase was
activated by treatment with 10 .mu.g/ml trypsin at 25.degree. C.
for 30 minutes. The activation reaction was stopped by the addition
of soybean trypsin inhibitor to a final concentration of 50
.mu.g/ml. Results were displayed by western blotting using the same
system as used for assaying protease resistance and are shown in
FIG. 6. The data indicate that collagen produced by expression of
DNA encoding .alpha.1(I) and .alpha.2(I) collagen lacking the N and
C propeptides is correctly folded and the monomer chains are
assembled in correct register.
[0060] The present invention has been detailed both by direct
description and by example. Equivalents and modifications of the
present invention will be apparent to those skilled in the art, and
are encompassed within the scope of the invention.
Sequence CWU 1
1
* * * * *