Note: Descriptions are shown in the official language in which they were submitted.
CA 02560161 2006-09-28
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TITLE OF THE INVENTION
[0001] PRODUCTION OF RECOMBINANT HUMAN COLLAGEN
FIELD OF THE INVENTION
[0002] The present invention relates to the production of polypeptides using
recombinant DNA systems. More specifically, the present invention is concerned
with
the production of human collagen using such systems.
BACKGROUND OF THE INVENTION
[0003] Collagen is the most abundant component of the extracellular matrix,
and is generally formed by the assembly of three polypeptide chains to create
a
trimeric structure. Nineteen different types of collagen have been described,
numbered as types I-XIX. For example, type I collagen, found in several
tissues such
as bone, tendons and skin, is a heterotrimeric molecule comprising two a-1 (I)
chains
and one a-2(I) chain. Type II collagen is a homotrimeric molecule comprising
three
a-1 (I I) chains. Type II I collagen, found in skin and vascular tissues, is a
homotrimeric
molecule comprising three a-1 (III) chains.
[0004] Various post-translational modifications during collagen biosynthesis
have been described, including, for example, the hydroxylation of proline
residues to
4-hydroxyproline by the enzyme prolyl 4-hydroxylase, and cleavage of N- and C-
propeptides of procollagen by N- and C-proteinase enzymes, respectively. Other
reported collagen post-translational enzymes include lysyl oxidase and lysyl
hydroxylase.
[0005] Human collagen is desirable for a number of therapeutic applications.
Its production by recombinant means is attractive for example to obtain
greater
amounts where insufficient amounts are available from natural sources, as well
as to
avoid any adverse immune reactions associated with the use of collagen from
non-
human sources.
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[0006] There is therefore a continued need to develop systems for the
recombinant production of collagen.
SUMMARY OF THE INVENT10N
[0007] The invention relates to the recombinant production of collagen.
[0008] More specifically, in accordance with the present invention, there is
provided a method for producing a recombinant human collagen polypeptide, said
method comprising: (a) culturing a host insect cell, wherein said insect cell
has been
infected, transfected or transformed with a recombinant baculovirus expression
vector
comprising: (i) a nucleotide sequence which encodes a collagen subunit,
operably
linked to a first promoter; and (ii) a nucleotide sequence which encodes a
collagen
post-translational enzyme or subunit thereof, operably linked to a second
promoter
different from said first promoter; and (b) recovering said collagen
polypeptide from
said host insect cell culture.
[0009] The invention further provides a method for producing a recombinant
human procollagen polypeptide, said method comprising: (a) culturing a host
insect
cell, wherein said insect cell has been infected, transfected or transformed
with a
recombinant baculovirus expression vector comprising: (i) a nucleotide
sequence
which encodes a collagen subunit, operably linked to a first promoter; and
(ii) a
nucleotide sequence which encodes a collagen post-translational enzyme or
subunit
thereof, operably linked to a second promoter; and (b) recovering the
procollagen
polypeptide from the host insect cell culture.
[0010] In an embodiment, the above-mentioned infected, transfected or
transformed host insect cell comprising the recombinant baculovirus expression
vector is obtained by a method comprising: (a) transfecting or transforming a
first host
insect cell with baculovirus DNA and an expression vector comprising: (i) a
nucleotide
sequence which encodes a collagen subunit, operably linked to a first
promoter; and
(ii) a nucleotide sequence which encodes a collagen post-translational enzyme
or
subunit thereof, operably linked to a second promoter; thereby to permit
integration of
CA 02560161 2006-09-28
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said expression vector into said baculovirus DNA to obtain a recombinant
baculovirus
expression vector; (b) isolating a nucleic acid molecule comprising the
recombinant
baculovirus expression vector from said host cell; and (c) transfecting or
transforming
a second host insect cell with the nucleic acid molecule obtained in (b)
thereby to
obtain an infected, transfected or transformed host insect cell comprising
said
recombinant baculovirus expression vector. In an embodiment, the method
further
comprises (d) culturing the infected, transfected or transformed host insect
cell
obtained in (c) under conditions suitable for production of recombinant
baculovirus;
(e) infecting a third host insect cell with the recombinant baculovirus
obtained in (d),
thereby to obtain an infected, transfected or transformed host insect cell
comprising
said recombinant baculovirus expression vector.
[0011] The invention further provides a recombinant collagen polypeptide
obtained by the above-mentioned method.
[0012] The invention further provides a recombinant procollagen polypeptide
obtained by the above-mentioned method.
[0013] The invention further provides a recombinant baculovirus expression
vector comprising: (i) a nucleotide sequence which encodes a collagen subunit,
operably linked to a first promoter; and (ii) a nucleotide sequence which
encodes a
collagen post-translational enzyme or subunit thereof, operably linked to a
second
promoter.
[0014] The invention further provides a host insect cell which has been
infected, transfected or transformed with the above-mentioned recombinant
baculovirus expression vector.
[0015] The invention further provides a method for producing a recombinant
human collagen or procollagen polypeptide, the method comprising: (a)
culturing a
host insect cell, wherein the insect cell has been infected, transfected or
transformed
with a recombinant baculovirus expression vector comprising: (i) a nucleotide
sequence which encodes a collagen subunit, operably linked to a p10 promoter;
and
CA 02560161 2006-09-28
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(ii) a nucleotide sequence which encodes a collagen post-translational enzyme
or
subunit thereof, operably linked to a poles promoter; and (b) recovering the
collagen or
procollagen polypeptide from said host insect cell culture.
[0016] In an embodiment, the above-mentioned first promoter is a p10
promoter, e.g., comprising the promoter region set forth in Figure 5 (SEQ ID
NO: 9).
[0017] In an embodiment, the above-mentioned second promoter is a
polyhedron (poles) promoter, e.g., comprising the promoter region set forth in
Figure 5
(SEQ ID NO: 10).
[0018] In an embodiment, the above-mentioned collagen subunit is a first
collagen subunit and wherein the recombinant baculovirus expression vector
further
comprises a nucleotide sequence which encodes a second collagen subunit,
operably
linked to a first promoter. In a further embodiment, the recombinant
baculovirus
expression vector further comprises a nucleotide sequence which encodes a
third
collagen subunit, operably linked to a first promoter.
[0019] In an embodiment, the above-mentioned subunit of a collagen post-
translational enzyme is a first subunit of a collagen post-translational
enzyme, and
wherein the recombinant baculovirus expression vector further comprises a
nucleotide sequence which encodes a second subunit of a collagen post-
translational
enzyme, operably linked to a second promoter.
[0020] In an embodiment, the above-mentioned collagen is selected from
collagen types I, II and III.
[0021] In an embodiment, the above-mentioned collagen is type II collagen
and the collagen subunit is a collagen a1 (II) subunit.
[0022] In an embodiment, the above-mentioned collagen is type III collagen
and the collagen subunit is a collagen a1 (III) subunit
CA 02560161 2006-09-28
[0023] In an embodiment, the above-mentioned collagen is type I collagen, the
first collagen subunit is a collagen a1 (I) subunit and the second collagen
subunit is a
collagen a2(1) subunit.
[0024] In an embodiment, the above-mentioned collagen post-translational
enzyme is selected from prolyl hydroxylase, lysyl oxidase and lysyl
hydroxylase. In a
further embodiment, the collagen post-translational enzyme is prolyl 4-
hydroxylase
[0025] In an embodiment, the above-mentioned collagen post-translational
enzyme is prolyl 4-hydroxylase and wherein the first subunit of a collagen
post-
translational enzyme is an alpha subunit of prolyl 4-hydroxylase and wherein
the
second subunit of a collagen post-translational enzyme is a beta subunit of
prolyl 4-
hydroxylase.
[0026] In a further aspect, the invention provides a method of increasing or
enhancing the purity of a collagen preparation, the method comprising
incubating the
collagen preparation under basic conditions such that the collagen is rendered
insoluble in the basic solution, and recovering the insoluble collagen. In an
embodiment, the method comprises dialyzing the collagen preparation against a
basic solution.
[0027] In a further aspect, the invention provides a method of preparing
collagen or processing a procollagen, the method comprising treating a
procollagen
sample with an elastase.
[0028] Other advantages and features of the present invention will become
more apparent upon reading of the following non-restrictive description of
specific
embodiments thereof, given by way of example only with reference to the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1: DNA (SEQ ID NO: 1) and polypeptide (SEQ ID NO: 2)
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sequences of human a-1(I) collagen (Homo sapiens colt-a1: Accession No.
NM 000088; GI: 14719826). SEQ ID NO: 2 corresponds to the coding sequence
defined by positions 127-4521 in SEQ ID NO: 1.
[0030] Figure 2: DNA (SEQ ID NO: 3) and polypeptide (SEQ ID NO: 4)
sequences of human a-2(I) collagen (Homo sapiens colll-a2: Accession No.
NM 000089, GI: 48762933), including the 5086 by sequence coding for pre-pro-
colll-a2 set forth in Accession No. 274616, GI: 1418929. SEQ ID NO: 4
corresponds
to the coding sequence defined by positions 472-4572 in SEQ ID NO: 3.
[0031] Figure 3: DNA (SEQ ID NO: 5) and polypeptide (SEQ ID NO: 6)
sequences of human prolyl 4-hydroxylase alpha subunit (a-p4H; Homo sapiens
p4Ha: 2722 by sequence set forth in Accession No. M24486 (clone PA-II), GI:
190785). SEQ ID NO: 6 corresponds to the coding sequence defined by positions
119-1723 in SEQ ID NO: 5.
[0032] Figure 4: DNA (SEQ ID NO: 7) and polypeptide (SEQ ID NO: 8)
sequences of human prolyl 4-hydroxylase beta subunit (~3-p4H; Homo sapiens
p4H~:
1956 by sequence set forth in Accession No. X05130, GI: 35654). SEQ ID NO: 8
corresponds to the coding sequence defined by positions 30-1556 in SEQ ID NO:
7
[0033] Figure 5: Sequences of Autographs californica multicapsid nuclear
polyhedrosis virus (AcMNPV) p10 (SEQ ID NO: 9) and poles (SEQ ID NO: 10)
promoter regions (derived from Autographs californica nucleopolyhedrovirus
complete genome Accession No. NC 001623, GI: 9627742), and also indicated in
technical materials for plasmid pBAC4x-1 T"~ (Novagen).
[0034] Figure 6. Photomicrographs taken under transmission electron
microscope of Sf9 insect cells transfected with recombinant baculovirus,
showing (A):
viral inclusions within the cytoplasm of the cells and (B): lysis of the cell
membrane by
the virus.
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[0035] Figure 7: Photomicrographs taken under phase contrast microscope of
the polymerized recombinant human type I collagen fibrils showing alignment
and
formation of a structured network, (40X). Panel A shows neo-formed recombinant
human type I collagen fibrils in formation, whereas panel B shows a sample
prepared
from the same batch and digested with pepsin, where no fibrils were detected.
[0036] Figure 8: Scanning electron microscopy image of polymerized
recombinant human type I collagen fibers, assembled naturally.
[0037] Figure 9: Results of SDS-PAGE (under reducing conditions) analysis
of recombinant human type I collagen chains, showing the purity of recombinant
human type I collagen and the expression of the a1- and a2-chains prepared
using
the method described herein.
[0038] Figure 10: Recombinant procollagen type I resolved by SDS-PAGE,
transferred on a nitrocellulose paper, followed by Concanavalin-A-biotin and
streptavidin-Peroxidase blotting, showing the positive staining of its
glycosylated
amino acid residues.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0039] Applicants describe herein a method of producing collagen using a
recombinant expression system. The method is preferably for the production of
human collagen.
[0040] Applicants have found that collagen may be successfully produced in a
recombinant expression system via the use of a single expression vector
comprising
both the nucleic acids) encoding a collagen subunit(s) and the nucleic acids)
encoding a collagen post-translational enzyme or subunit(s) thereof. Prior to
applicants' studies described herein, attempts to produce recombinant collagen
entailed the use of multiple expression vectors.
CA 02560161 2006-09-28
[0041] In an embodiment, four nucleic acids (which encode a corresponding
polypeptide) may be inserted into a single vector, preferably a baculovirus
vector. For
example, these four nucleic acids may encode a first collagen subunit, a
second
collagen subunit, a first subunit of a collagen post-translational enzyme, and
a second
subunit of a collagen post-translational enzyme, respectively.
[0042] In a preferred embodiment, the expression system is a
baculovirus/insect cell expression system. This system is advantageous because
it
provides, among other things, high levels of expression of the recombinant
protein
with the appropriate post-translational modifications and is amenable to scale-
up for
large-scale production. Various reagents for baculovirus/insect cell
expression
systems are known in the art and are commercially available. The Baculovirus
Expression Vector System (BEVS) is one of the most powerful and versatile
eukaryotic expression systems available. This expression system relies on the
generation of recombinant baculoviruses in which viral genes, not essential
for viral
replication in cell culture, are replaced by DNA sequences of interest,
(O'Reilly et al.,
1992; Kidd and Emery, 1993). The recombinant viral DNA is typically
transfected into
Spodoptera frugiperda (Sf9) insect cells, clonal derivative of the fall
armyworm
Spodoptera frugiperda ovarian cell line, IPLB-Sf21-AE, (Sf21), (Vaughn et al,
1977;
Nobiron et al, 2003). The recombinant proteins expressed in the baculovirus
system
are properly folded, disulphide bonded, oligomerized, and localized in the
same
subcellular compartment as the authentic protein (Kidd and Emery, 1993).
Insect cells
are also capable of performing several post-translational modifications such
as N-
and O- glycosylation, phosphorylation, acylation, amidation,
carboximethilation, signal
peptide cleavage, and proteolytic cleavage (Matsuura et al., 1987; Nokelainen
M.,
2000). The sites where these modifications occur are often identical to those
of the
authentic protein in its native cellular environment (Hoss et al., 1990; Kloc
et al.,
1991; Kuroda et al., 1990). In addition, insect cells possess a low prolyl-4-
hydroxilase
activity (Veijola et al., 1994). In this system, expression of the above-noted
nucleic
acids may be driven by the polyhedron (poles) promoter or the p10 promoter,
both of
which are known for use in baculovirus expression systems. Multiple copies of
these
promoters may be used in a vector to express multiple nucleic acids of
interest.
[0043] In a preferred embodiment, the expression of the above-noted nucleic
CA 02560161 2006-09-28
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acids is driven by two different promoters, e.g., respective first and second
promoters,
such as the poles promoter for the expression of the collagen subunit and the
p10
promoter for the expression of the collagen post-translational enzyme or
subunit
thereof.
[0044] Accordingly, in a first aspect, the invention provides a method for
producing a recombinant collagen or procollagen polypeptide, such as a human
collagen or procollagen polypeptide, said method comprising:
(a) culturing a host cell, such as a host insect cell, wherein the host cell
has been
infected, transfected or transformed with a recombinant expression vector
(e.g., a recombinant baculovirus expression vector) comprising:
(i) a nucleotide sequence which encodes a collagen subunit, operably linked
to a first promoter (e.g., a polyhedron (poles) promoter); and
(ii) a nucleotide sequence which encodes a collagen post-translational enzyme
or subunit thereof, operably linked to a second promoter (e.g. a p10
promoter);
(b) recovering said collagen or procollagen polypeptide from said host cell
culture.
[0045] In embodiments, the host cell is a eukaryotic host cell selected from
an
insect cell, a fungal (e.g. yeast) cell, a mammalian cell and a plant cell. In
a preferred
embodiment the host cell is an insect host cell, such as a Spodoptera
frugiperda-
derived cell (e.g., Sf9 or Sf21 ).
[0046] In embodiments, single or multiple collagen or procollagen subunits can
be expressed using the method of the invention. Therefore, in embodiments, the
vector further comprises a nucleotide sequence which encodes a second collagen
subunit operably linked to a promoter (e.g., a poles or p10 promoter). In a
further
embodiment, the vector yet further comprises a nucleotide sequence which
encodes
a third collagen subunit operably linked to a promoter (e.g., a poles or a p10
promoter).
[0047] In an embodiment, the vector further comprises a nucleotide sequence
which encodes a further subunit of a collagen post-translational enzyme,
operably
CA 02560161 2006-09-28
linked to a promoter (e.g., a poles or a p10 promoter).
[0048] In embodiments, the collagen is selected from collagen types I-XIX.
The appropriate collagen subunits for expression in each case are known in the
art.
For example, relevant subunits in respect of certain types of collagen are set
forth in
Table 1.
[0049] Table 1. Structures of certain types of collagen
T Structure
a
I heterotrimeric:two a 1 I chains; one a 2 I chain
II homotrimeric:three a 1 (I I) chains
III homotrimeric:three a 1 III chains
IV most common form:
heterotrimeric:two a 1 IV) chains; one a 2 IV chain
V multiple e.g.:
forms, two a 1 (V) chains; one a 2(V) chain
heterotrimeric:one each of a 1 (V), a 2(V) and a 3(V)
heterotrimeric:chains
homotrimeric:three a 1 (V) chains
VI heterotrimeric:one each of a 1 VI , a 2 VI and a 3
VI chains
VII homotrimeric:three a 1 VII chains
VII heterotrimeric:
I two a 1
(VII I)
chains;
one a 2(V1
II) chain
(other
structures
also described
IX heterotrimeric:one each of a 1 (IX , a 2 IX) and a
3 IX) chains
X homotrimeric:three a 1 (X chains
XI heterotrimeric:one each of a 1 XI , a 2 XI and a 3
XI chains
XII homotrimeric:three a 1 (XI I chains
XIV homotrimeric:three a 1 (XIV) chains
[0050] In embodiments, the collagen is selected from types I, II and III. In
the
case of type II collagen, the vector comprises a nucleotide sequence which
encodes
a collagen a1 (II) subunit, operably linked to a promoter (e.g., a poles or a
a p10
promoter). In the case of type III collagen, the vector comprises a nucleotide
sequence which encodes a collagen a1 (Ill) subunit, operably linked to a
promoter
(e.g., a poles or a p10 promoter). In the case of type I collagen the vector
comprises a
nucleotide sequence which encodes a collagen a1 (I) subunit, operably linked
to a
promoter (e.g., a poles or a p10 promoter), and further comprises a a
nucleotide
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sequence which encodes a collagen a2(1) subunit, operably linked to a promoter
(e.g., a poles or a p10 promoter).
[0051] In further embodiments, multiple copies of a a nucleotide sequence
which encodes a collagen subunit may be included in the vector. For example,
in the
case of type II collagen (which has a homotrimeric structure), two (or more)
copies of
a nucleotide sequence encoding a collagen a1 (II) subunit may be included in
the
vector.
[0052] An example of a human collagen a1 (I) subunit corresponds to the
polypeptide (SEQ ID NO: 2) set forth in Figure 1, which also sets forth the
nucleic
acid sequence (SEQ ID NO: 1) encoding the polypeptide. Positions 127-192 of
SEO
ID NO: 1 correspond to the signal peptide. Positions 193-4518 of SEQ ID NO: 1
correspond to the proprotein.
[0053] An example of a human collagen a2(1) subunit corresponds to the
polypeptide (SEQ ID NO: 4) set forth in Figure 2, which also sets forth the
nucleic
acid sequence (SEQ ID NO: 3) encoding the polypeptide.
[0054] In embodiments, the collagen post-translational enzyme is selected
from prolyl hydroxylase (e.g. prolyl 4-hydroxylase), lysyl oxidase, lysyl
hydroxylase,
N-proteinase and C-proteinase.
[0055] Prolyl 4-hydroxylase is classified under enzyme classification EC
1.14.11.2 and catalyzes the hydroxylation of proline residues to 4-
hydroxyproline in
the synthesis of collagen or procollagen. The human form comprises two
subunits,
denoted as alpha and beta subunits. The human alpha-I isoform of the alpha
subunit
corresponds to for example Genbank accession No. M24486. The human alpha-II
isoform of the alpha subunit corresponds to for example Genbank accession No.
U90441. The human beta subunit corresponds to for example Genbank accession
No. X05130. The vertebrate enzyme is a tetramer comprising two alpha and two
beta
subunits.
CA 02560161 2006-09-28
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[0056] An example of a human prolyl 4-hydroxylase alpha subunit
corresponds to the polypeptide (SEQ ID NO: 6) set forth in Figure 3, which
also sets
forth the nucleic acid sequence (SEQ ID NO: 5) encoding the polypeptide.
[0057] An example of a prolyl 4-hydroxylase beta subunit corresponds to the
polypeptide (SEQ ID NO: 8) set forth in Figure 4, which also sets forth the
nucleic
acid sequence (SEQ ID NO: 7) encoding the polypeptide. Positions 30-80 of SEQ
ID
NO: 7 correspond to the signal peptide.
[0058] Lysyl hydroxylase is classified under classification EC 1.14.11.4 and
catalyzes the hydroxylation of Lys residues in the -X-Lys-Gly- triplet motif
of
collagens. The enzyme is a homodimer of two alpha subunits. Various forms of
the
human enzyme correspond to for example Swiss-Prot accession Nos. Q02809,
000469 and 060568.
[0059] In the studies described herein, applicants have determined that post-
translational processing of collagen may be effected by treatment with
elastase.
Therefore, in a further aspect, the invention provides a method of preparing
collagen
or processing a procollagen, said method comprising treating said procollagen
sample with an elastase enzyme.
[0060] A preferred expression system to be used in the method of the
invention is the baculovirus/insect cell expression system, whereby the
expression
vector comprising the above-noted nucleic acids is introduced into a host
insect cell
using a baculovirus construct. The host cell is thus cultured under conditions
suitable
for polypeptide production. An example of such a system utilizes the
Autographica
californica nuclear polyhydrosys virus (AcNPV), which grows in Spodoptera
frugiperda cells. The nucleic acids) encoding the recombinant polypeptide(s)
of
interest (operably linked to an appropriate promoter for expression in the
host cell)
can be inserted into a non-essential region of AcNPV such as the polyhedron
gene. In
an embodiment, recombination of these nucleic acids into the non-essential
region
can result in the replacement or disruption of a marker gene, such as the lacz
gene
(~-galactosidase), thus allowing selection of recombinants based on the
absence of
CA 02560161 2006-09-28
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the marker's activity. Such selection is sometimes referred to as a "plaque
assay", as
plaques may be selected on the basis of the absence or presence of marker
activity.
Such recombinant viruses may be used to infect the host insect cell for
expression of
the polypeptide(s) encoded by the inserted nucleic acid(s).
[0061] Preparation of such recombinant viruses typically entails the co-
infection of linear viral DNA and a vector comprising the nucleic acids)
encoding the
recombinant polypeptide(s) of interest (operably linked to an appropriate
promoter for
expression in the host cell) into a host insect cell, whereby recombination
results in
the insertion of the nucleic acids) into the viral DNA. Recombinant virus
produced
may be identified by plaque assay, which is typically repeated to perform a
second
round of plaque purification. Ultimately, a stock of recombinant virus is
obtained and
used to infect host insect cells for polypeptide expression.
[0062] In an embodiment, the method comprises an isolation or amplification
step, whereby the recombinant viral DNA comprising the nucleic acids) encoding
the
polypeptide(s) of interest (operably linked to an appropriate promoter for
expression
in the host cell) is isolated or obtained by amplification (e.g. by polymerase
chain
reaction [PCR]). The isolated or amplified recombinant viral DNA may then be
directly introduced (e.g. transfected or transformed) into a host insect cell.
Applicants
have found that the use of such an additional isolation or amplification step
further
allows for the efficient preparation of host insect cells for the production
of
recombinant collagen or procollagen, rather than relying on baculovirus
infection
alone. Recombinant virus obtained from, for example, the culture medium of
such
host insect cells may be used as a viral stock to infect other host insect
cells for
further recombinant polypeptide production.
[0063] Accordingly, in an embodiment, the above-mentioned infected,
transfected or transformed host insect cell comprising said recombinant
baculovirus
expression vector is obtained by a method comprising:
(a) transfecting or transforming a first host insect cell with baculovirus DNA
and an
expression vector comprising: (i) a nucleotide sequence which encodes a
collagen
CA 02560161 2006-09-28
14
subunit, operably linked to a promoter (e.g., a poles promoter); and (ii) a
nucleotide
sequence which encodes a collagen post-translational enzyme or subunit
thereof,
operably linked to a promoter (e.g., a p10 promoter); thereby to permit
integration of
said expression vector into said baculovirus DNA to obtain a recombinant
baculovirus
DNA expression vector;
(b) isolating a nucleic acid molecule comprising said recombinant baculovirus
DNA
expression vector from said host cell; and
(c) transfecting or transforming a second host insect cell with said nucleic
acid
molecule obtained in (b) thereby to obtain an infected, transfected or
transformed
host insect cell comprising said recombinant baculovirus expression vector.
[0064] In a further embodiment, the just-noted method further comprises: (d)
culturing said infected, transfected or transformed host insect cell obtained
in (c)
above under conditions suitable for production of recombinant baculovirus; and
(e)
infecting a third host insect cell with the recombinant baculovirus obtained
in (d)
above, thereby to obtain an infected, transfected or transformed host insect
cell
comprising said recombinant baculovirus expression vector.
[0065] The invention further provides a recombinant collagen or procollagen
polypeptide obtained by the above-mentioned method.
[0066] The invention further provides the above-mentioned recombinant
expression vector. In an embodiment, the expression vector is a recombinant
baculovirus DNA expression vector.
[0067] The invention further provides a host cell, such as an insect host
cell,
which has been infected, transfected or transformed with the above-mentioned
recombinant viral DNA expression vector.
[0068] "p10 promoter" as used herein refers to a nucleic acid sequence
derived from the Autographs californica multicapsid nuclear polyhedrosis virus
CA 02560161 2006-09-28
(AcMNPV) which can modulate the transcription of the AcMNPV p10 gene. Details
of
the p10 promoter are set forth in Autographs californica nucleopolyhedrovirus
complete genome Accession No. NC 001623, GI: 9627742, as well as in the
technical materials for plasmid pBAC4x-1 T"" (Novagen).
[0069] "poles promoter" or "polyhedron promoter" as used herein refers to a
nucleic acid sequence derived from the AcMNPV which can modulate the
transcription of the AcMNPV polyhedron gene. Details of the poles promoter are
set
forth in Autographs californica nucleopolyhedrovirus complete genome Accession
No.
NC 001623, GI: 9627742, as well as in the technical materials for plasmid
pBAC4x-
1 T"" (Novagen).
[0070] "Collagen" as used herein refers to any of the known collagen types (I-
XIX) as well as any variants as described herein, and includes single chain,
heterotrimeric and homotrimeric molecules of collagen. "Procollagen" as used
herein
is similarly defined and refers to any of the known procollagen as well as any
variants
as described herein, and includes single chain, heterotrimeric and
homotrimeric
molecules of procollagen, but differs from collagen in that it additionally
comprises N-
terminal and/or C-terminal peptides which are cleaved off for example by N-
proteinase and/or C-proteinase enzymes.
[0071] As noted above, an isolated nucleic acid, for example a nucleic acid
sequence encoding a polypeptide of the invention (e.g., a collagen or
procollagen
subunit; a collagen post-tranlational enzyme or subunit thereof), or homolog,
fragment
or variant thereof, may further be incorporated into a vector, such as a
recombinant
expression vector. In an embodiment, the vector will comprise transcriptional
regulatory sequences or a promoter operably linked to a nucleic acid
comprising a
sequence capable of encoding a peptide compound, polypeptide or domain of the
invention. A first nucleic acid sequence is "operably linked" with a second
nucleic
acid sequence when the first nucleic acid sequence is placed in a functional
relationship with the second nucleic acid sequence. For instance, a promoter
is
operably linked to a coding sequence if the promoter affects the transcription
or
expression of the coding sequences. Generally, operably linked DNA sequences
are
CA 02560161 2006-09-28
16
contiguous and, where necessary to join two protein coding regions, in reading
frame.
However, since for example enhancers generally function when separated from
the
promoters by several kilobases and intronic sequences may be of variable
lengths,
some polynucleotide elements may be operably-linked but not contiguous.
"Transcriptional regulatory sequence/element" is a generic term that refers to
DNA
sequences, such as initiation and termination signals, enhancers, and
promoters,
splicing signals, polyadenylation signals which induce or control
transcription of
protein coding sequences with which they are operably-linked. "Promoter"
refers to a
DNA regulatory region capable of binding directly or indirectly to RNA
polymerase in a
cell and initiating transcription of a downstream (3' direction) coding
sequence. For
purposes of the present invention, the promoter is bound at its 3' terminus by
the
transcription initiation site and extends upstream (5' direction) to include
the minimum
number of bases or elements necessary to initiate transcription at levels
detectable
above background. Within the promoter will be found a transcription initiation
site
(conveniently defined by mapping with S1 nuclease), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA" boxes and
"CCAT"
boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to
the -
and -35 consensus sequences.
[0072] As noted above, the invention relates to the recombinant production of
collagen or procollagen. Thus, various nucleic acid sequences of the invention
may
be recombinant sequences. The term "recombinant" means that something has been
recombined, so that when made in reference to a nucleic acid construct the
term
refers to a molecule that is comprised of nucleic acid sequences that are
joined
together or produced by means of molecular biological techniques. The term
"recombinant" when made in reference to a protein or a polypeptide refers to a
protein or polypeptide molecule which is expressed using a recombinant nucleic
acid
construct created by means of molecular biological techniques. The term
"recombinant" when made in reference to genetic composition refers to a gamete
or
progeny or cell or genome with new combinations of alleles that did not occur
in the
parental genomes. Recombinant nucleic acid constructs may include a nucleotide
sequence which is ligated to, or is manipulated to become ligated to, a
nucleic acid
sequence to which it is not ligated in nature, or to which it is ligated at a
different
CA 02560161 2006-09-28
17
location in nature. Referring to a nucleic acid construct as 'recombinant'
therefore
indicates that the nucleic acid molecule has been manipulated using genetic
engineering, i.e., by human intervention. Recombinant nucleic acid constructs
may
for example be introduced into a host cell by transformation. Such recombinant
nucleic acid constructs may include sequences derived from the same host cell
species or from different host cell species, which have been isolated and
reintroduced
into cells of the host species. Recombinant nucleic acid construct sequences
may
become integrated into a host cell genome, either as a result of the original
transformation of the host cells, or as the result of subsequent recombination
and/or
repair events.
[0073] The recombinant polypeptides of the invention may also be expressed
in the form of a suitable fusion protein, comprising an amino acid sequence of
a
polypeptide of the invention linked to further polypeptide sequence (e.g. a
heterologous sequence). Such fusion proteins are typically produced by
expression
of recombinant nucleic acids encoding them. In embodiments, the further
polypeptide
sequence may confer various functions such as to facilitate cellular
localization/secretion, detection and purification (e.g. via affinity
methods).
[0074) The terminology "amplification pair" refers herein to a pair of
oligonucleotides (oligos), which are selected to be used together in
amplifying a
selected nucleic acid sequence by one of a number of types of amplification
processes, preferably a polymerase chain reaction. Other types of
amplification
processes include ligase chain reaction, strand displacement amplification, or
nucleic
acid sequence-based amplification. As commonly known in the art, the oligos
are
designed to bind to a complementary sequence under selected conditions.
[0075] Oligonucleotide probes or primers of the present invention may be of
any suitable length, depending on the particular assay format and the
particular
needs and targeted sequences employed. In general, the oligonucleotide probes
or
primers are at least 12 nucleotides in length, preferably between 15 and 24
molecules, and they may be adapted to be especially suited to a chosen nucleic
acid
amplification system. As commonly known in the art, the oligonucleotide probes
and
CA 02560161 2006-09-28
18
primers can be designed by taking into consideration the melting point of
hybrizidation
thereof with its targeted sequence (see below and in Sambrook et al., 1989,
Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories;
Ausubel et
al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc.,
N.Y.).
[0076] "Homology" and "homologous" refers to sequence similarity between
two peptides or two nucleic acid molecules. Homology can be determined by
comparing each position in the aligned sequences. A degree of homology between
nucleic acid or between amino acid sequences is a function of the number of
identical
or matching nucleotides or amino acids at positions shared by the sequences.
As the
term is used herein, a nucleic acid sequence is "homologous" to another
sequence if
the two sequences are substantially identical and the functional activity of
the
sequences is conserved (as used herein, the term 'homologous' does not infer
evolutionary relatedness). Two nucleic acid or polypeptide sequences are
considered
"substantially identical" if, when optimally aligned (with gaps permitted),
they share at
least about 50% sequence similarity or identity, or if the sequences share
defined
functional motifs. In alternative embodiments, sequence similarity in
optimally aligned
substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90%
or
95%. As used herein, a given percentage of homology between sequences denotes
the degree of sequence identity in optimally aligned sequences. Similarly,
"substantially complementary" nucleic acids are nucleic acids in which the
complement of one molecule is "substantially identical" to the other molecule.
An
"unrelated" or "non-homologous" sequence shares less than 40% identity, though
preferably less than about 25 % identity, with a nucleic acid or polypeptide
of the
invention.
[0077] Alignment of sequences for comparisons of identity may be conducted
using a variety of algorithms and methods, such as those of Smith and Waterman
(1981, Adv. Appl. Math 2: 482), Needleman and Wunsch (1970, J. Mol. Biol.
48:443),
Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85: 2444), and the
computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA
and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, Madison, WI, U.S.A.). Sequence identity may also be determined using
the
BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10
(using
CA 02560161 2006-09-28
19
the published default settings). Software for performing BLAST analysis may be
available through the National Center for Biotechnology Information (through
the
Internet at http://www.ncbi.nlm.nih.giov/). The BLAST algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short words of
length W
in the query sequence that either match or satisfy some positive-valued
threshold
score T when aligned with a word of the same length in a database sequence. T
is
referred to as the neighbourhood word score threshold. Initial neighbourhood
word
hits act as seeds for initiating searches to find longer HSPs. The word hits
are
extended in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extension of the word hits in each direction
is
halted when the following parameters are met: the cumulative alignment score
falls
off by the quantity X from its maximum achieved value; the cumulative score
goes to
zero or below, due to the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST algorithm
parameters W, T and X determine the sensitivity and speed of the alignment.
The
BLAST program may use as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89:
10915-
10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or
0.001 or
0.0001 ), M=5, N=4, and a comparison of both strands. One measure of the
statistical
similarity between two sequences using the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability by which a
match
between two nucleotide or amino acid sequences would occur by chance. In
alternative embodiments of the invention, nucleotide or amino acid sequences
are
considered substantially identical if the smallest sum probability in a
comparison of
the test sequences is less than about 1, preferably less than about 0.1, more
preferably less than about 0.01, and most preferably less than about 0.001.
(0078] An alternative indication that two nucleic acid sequences are
substantially complementary is that the two sequences hybridize to each other
under
moderately stringent, or preferably stringent, conditions. "Nucleic acid
hybridization"
or "hybridize" generally refer to the hybridization of two single-stranded
nucleic acid
molecules having complementary base sequences, which under appropriate
conditions will form a thermodynamically favored double-stranded structure.
Examples of hybridization conditions can be found in the two laboratory
manuals
CA 02560161 2006-09-28
referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989, supra)
and
are commonly known in the art. In the case of a hybridization to a
nitrocellulose filter,
as for example in the well known Southern blotting procedure, a nitrocellulose
filter
can be incubated overnight at 65°C with a labeled probe in a solution
containing 50%
formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1 % SDS,
and
100 pg/ml denatured carrier DNA (i.e. salmon sperm DNA). The non-specifically
binding probe can then be washed off the filter by several washes in 0.2 x
SSC/0.1
SDS at a temperature which is selected in view of the desired stringency: room
temperature (low stringency), 42°C (moderate stringency) or 65°C
(high stringency).
The selected temperature is based on the melting temperature (Tm) of the DNA
hybrid (Sambrook et al. 1989, supra). Of course, RNA-DNA hybrids can also be
formed and detected. In such cases, the conditions of hybridization and
washing can
be adapted according to well known methods by the person of ordinary skill.
Stringent
conditions will be preferably used (Sambrook et a1.,1989, supra).
[0079] As used herein, a "primer" defines an oligonucleotide which is capable
of annealing to a target sequence, thereby creating a double stranded region
which
can serve as an initiation point for DNA synthesis under suitable conditions.
[0080] Amplification of a selected, or target, nucleic acid sequence may be
carried out by a number of suitable methods. See generally Kwoh et al., 1990,
Am.
Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been
described
and can be readily adapted to suit particular needs of a person of ordinary
skill. Non-
limiting examples of amplification techniques include polymerase chain
reaction
(PCR), ligase chain reaction (LCR), strand displacement amplification (SDA),
transcription-based amplification, the Q- replicase system and NASBA (Kwoh et
al.,
1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988,
BioTechnology
6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook
et
al., 1989, supra). Preferably, amplification will be carried out using PCR.
[0081] Polymerase chain reaction (PCR) is carried out in accordance with
known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;
and
4,965,188. In general, PCR involves a treatment of a nucleic acid sample
(e.g., in the
CA 02560161 2006-09-28
21
presence of a heat stable DNA polymerase) under hybridizing conditions, with
one
oligonucleotide primer for each strand of the specific sequence to be
detected. An
extension product of each primer which is synthesized is complementary to each
of
the two nucleic acid strands, with the primers sufficiently complementary to
each
strand of the specific sequence to hybridize therewith. The extension product
synthesized from each primer can also serve as a template for further
synthesis of
extension products using the same primers. Following a sufficient number of
rounds
of synthesis of extension products, the sample is analysed to assess whether
the
sequence or sequences to be detected are present. Detection of the amplified
sequence may be carried out by visualization following Etl3r staining of the
DNA
following gel electrophoresis, or using a detectable label in accordance with
known
techniques, and the like. For a review on PCR techniques (see PCR Protocols, A
Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
[0082] Ligase chain reaction (LCR) is carried out in accordance with known
techniques (Weiss, 1991, Science 254:1292). Adaptation of the protocol to meet
desired needs can be carried out by a person of ordinary skill. Strand
displacement
amplification (SDA) is also carried out in accordance with known techniques or
adaptations thereof to meet the particular needs (Walker et al., 1992, Proc.
Natl.
Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).
[0083] The term "vector" is commonly known in the art and defines a plasmid
DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into
which DNA of the present invention can be cloned. Numerous types of vectors
exist
and are well known in the art.
[0084] The term "expression" defines the process by which a gene is
transcribed into mRNA (transcription), the mRNA is then translated
(translation) into
one polypeptide (or protein) or more.
[0085] The recombinant expression vector of the present invention can be
constructed by standard techniques known to one of ordinary skill in the art
and
found, for example, in Sambrook et al. (supra). A variety of strategies are
available
CA 02560161 2006-09-28
22
for ligating fragments of DNA, the choice of which depends on the nature of
the
termini of the DNA fragments and can be readily determined by persons skilled
in the
art. The vectors of the present invention may also contain other sequence
elements
to facilitate vector propagation and selection in bacteria and host cells. In
addition,
the vectors of the present invention may comprise a sequence of nucleotides
for one
or more restriction endonuclease sites. Coding sequences such as for
selectable
markers and reporter genes are well known to persons skilled in the art.
[0086] A recombinant expression vector comprising a nucleic acid sequence
of the present invention may be introduced into a host cell, which may include
a living
cell capable of expressing the protein coding region from the defined
recombinant
expression vector. The living cell may include both a cultured cell and a cell
within a
living organism. Accordingly, the invention also provides host cells
containing the
recombinant expression vectors of the invention. The terms "host cell" and
"recombinant host cell" are used interchangeably herein. Such terms refer not
only to
the particular subject cell but to the progeny or potential progeny of such a
cell.
Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to
the parent cell, but are still included within the scope of the term as used
herein.
[0087] Vector DNA can be introduced into cells via conventional
transformation or transfection techniques. The terms "transformation" and
"transfection" refer to techniques for introducing foreign nucleic acid into a
host cell,
including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-
mediated transfection, lipofection, electroporation, microinjection and viral-
mediated
transfection. Suitable methods for transforming or transfecting host cells can
for
example be found in Sambrook et al. (supra), and other laboratory manuals.
"Infection" as used herein refers to the introduction of nucleic acids into a
cell using a
virus or viral vector, such as a baculovirus.
[0088] Polypeptides produced by the recombinant methods described herein
can be purified according to standard protocols that take advantage for
example of
the intrinsic properties thereof, such as size and charge (i.e. SDS gel
electrophoresis,
CA 02560161 2006-09-28
23
gel filtration, dialysis, centrifugation, ion exchange chromatography...). In
addition,
the recombinant polypeptide can be purified via affinity chromatography using
polyclonal or monoclonal antibodies or other affinity-based systems (e.g.
using a
suitable incorporated "tag" in the form of a fusion protein and its
corresponding
ligand). Its structure can be further modified using one or more enzymes or
bioactive
compounds.
[0089] Applicants have further demonstrated herein that dialysis under basic
conditions allows for the efficient purification of collagen. As described in
the
Examples below, when dialyzed under basic conditions (e.g., at about pH 8.5
[e.g., in
a sodium acetate buffer]), assembled collagen fibrils polymerize while most
contaminant proteins are solubilized. The collagen can then be recovered by
appropriate means (e.g., centrifugation).
[0090] Accordingly, in a further aspect, the invention provides a method of
enhancing the purity of a collagen preparation, said method comprising
incubating the
collagen under basic conditions (e.g., dialyzing the preparation against a
basic
solution), and recovering the collagen by suitable means (e.g.,
centrifugation,
filtration, etc.). "Basic conditions" as used herein refers to conditions
exhibiting an
average pH greater than ph 7Ø In an embodiment, the pH is greater than or
equal to
7.5, in a further embodiment, greater than or equal to 8Ø In an embodiment,
the pH
is about 8.5. Various buffer systems (e.g. acetate) are known in the art to
prepare
solutions exhibiting such basic conditions.
[0091] In a further embodiment a product of the invention (e.g., a polypeptide
[e.g., a collagen polypeptide]) is substantially pure. A compound is
"substantially
pure" when it is separated from the components that naturally accompany it.
Typically, a compound is substantially pure when it is at least 60%, more
generally
75% or over 90%, by weight, of the total material in a sample. Thus, for
example, a
polypeptide that is chemically synthesised or produced by recombinant
technology
will generally be substantially free from its naturally associated components.
A
nucleic acid molecule is substantially pure when it is not immediately
contiguous with
(i.e., covalently linked to) the coding sequences with which it is normally
contiguous in
CA 02560161 2006-09-28
24
the naturally occurring genome of the organism from which the DNA of the
invention
is derived. A substantially pure compound can be obtained, for example, by
extraction from a natural source; by expression of a recombinant nucleic acid
molecule encoding a polypeptide compound; or by chemical synthesis. Purity can
be
measured using any appropriate method such as column chromatography, gel
electrophoresis, HPLC, etc.
[0092] A homolog, variant and/or fragment of a polypeptide of the invention
which retains activity, and nucleic acids encoding such a homolog, variant
and/or
fragment, may also be used in the methods of the invention. Homologs include
polypeptide sequences, which are substantially identical to the amino acid
sequence
of a a polypeptide of the invention, sharing significant structural and
functional
homology with a polypeptide of the invention. Variants include, but are not
limited to,
polypeptides, which differ from a a polypeptide of the invention by any
modifications,
and/or amino acid substitutions, deletions or additions. Modifications can
occur
anywhere including the polypeptide backbone, (i.e. the amino acid sequence),
the
amino acid side chains and the amino or carboxy termini. Such substitutions,
deletions or additions may involve one or more amino acids. Fragments include
a
fragment or a portion of a polypeptide of the invention, or a fragment or a
portion of a
homolog or variant of a polypeptide of the invention.
[0093] The present invention is illustrated in further details by the
following
non-limiting examples.
EXAMPLES
[0094] Example 1: Materials and methods
[0095] pBAC4x-1 T"" transfer plasmid and Insect GeneJuiceT"' transfection
reagent were obtained from Novagen (EMD Biosciences / Novagen /VWR CANLAB,
Mississaga, Ontario, Canada). Clones containing nucleic acids encoding
subunits of
collagen and prolyl-4-hydroxylase were obtained from American Type Culture
Collection (ATCC) as follows: ATCC # 59480 for P4H beta subunit; ATCC # 138677
CA 02560161 2006-09-28
for P4H alpha subunit; ATCC # 95501 for coil I alpha-2 chain; and ATCC # 95499
for
colt I alpha-1 chain.
[0096] Example 2: Preparation of baculovirus expression construct.
(0097] Preparation of the baculovirus expression construct pBacNl-hcoll I was
performed by modification of pBAC4x-1 T"'. The ~ subunit of P4H was inserted
first. It
was cloned and the cDNA was amplified by PCR with primers tagged to the EcoRl/
Spel ends. The PCR amplified cDNA was ligated to the sites Smal and Spel in
linearized pBAC4x-1 T"'. Secondly, the coil a-1 (I) subunit was inserted via
the insertion
of an Xbal restriction enzyme fragment containing DNA encoding the coll a-1
(I)
subunit into the Xbal site of linearized pBAC4x-1 T"". Thirdly, The colt a-
2(I) subunit
cDNA was inserted via the insertion of an Sphl restriction enzyme fragment
containing DNA encoding the coil a-2(I) subunit into the Sphl site of the
linearized
pBAC4x-1 T"~. Subsequently, the a and ~3 subunits of P4H were inserted. The
detailed
sequence of the final construct, is desribed in Table 2.
[0098] Table 2: Description of pBacNl-hcoll I sequence (18,169 bps)
Sequence Description
1-1242 lasmid Bac4x derived se uence
1243-1248 Bglll site
1249-3004 P4H sequence (oriented counterclockwise)
3148-3152 B III site
3153-3401 plasmid Bac4x derived sequence
3402-3406 Xbal site
3408-7207 Col1 a1 (I) sequence and non coding
se uence (oriented counterclockwise)
7208-7213 Xbal site
7214-7453 lasmid pBac4x derived sequence
7454-7459 Smal site
7480-9578 P4Ha se uence
9579-9584 Spel site
9585-9774 lasmid Bac4x derived se uence
9775-9780 Hindlll site
14255-14260 S hl site
14261-18169 lasmid Bac4x derived se uence
CA 02560161 2006-09-28
26
[0099] Example 3: Expression, maturation and purification of recombinant
human collagen.
[00100] Infected Sf9 cells were grown for 2-6 days in Grace's medium
supplemented with ascorbic acid (50 ug/ml) to stimulate collagen synthesis.
sf9 cells
(suspension of 1 L) were centrifuged to obtain a pellet of cells that contain
human
recombinant procollagen. The cell pellet was resuspended in about 100 ml of 50
mM
Tris-HCI buffer containing 0.2 M NaCI, at pH 7.4. The cells were broken
mechanically
to liberate pro-collagen (e.g. freezing-thawing twice at -20°C and
4°C, respectively).
Since the extract also contains DNA, coming out of the broken cells, that can
provoke
DNA-pro-collagen aggregates, DNAse treatment was used to eliminate the DNA.
[00101] The procoll / colt suspension was then digested with elastase at
4°C,
for 2-3 hrs by adding half volume of 50 mM Tris-HCL, pH 8.5 containing
elastase (1-2
mg/ml). After this incubation period, the assembled collagen fibrils were
dialyzed
against a sodium acetate buffer pH 8.5 for 72 hrs at 4°C. The white
fibrils polymerize,
while most contaminant proteins were solubilized during dialysis.
[00102] After the completion of the dialysis, the collagen was centrifuged for
20
min at 6000 rpm and the pellet was rinsed twice with megapure water,
centrifuging
each time to recuperate the pellet. A protease inhibitor cocktail was added to
the
fibrils. The collagen was solubilized in citric acid 0.075M, pH 3.7,
overnight, and the
residual contaminant proteins that are precipitated were discarded by
centrifugation
(pellet). The supernatant, containing the acid-solubilized collagen, was
dialysed
against phosphate buffer 0.02M, pH 9.2 to 9.5, at 4°C. The fibrils
slowly precipitated
within 2-3 days. The fibrils were centrifuged, washed 3 times and resuspended
in
megapure water. The suspension was frozen at -86°C and lyophilized.
[00103] In some experiments, the procoll / colt suspension was then
precipitated with ammonium sulfate for about 2 hrs at 4°C and
centrifuged to obtain a
pellet of proteins. The pellet was resuspended in Tris-HCI buffer containing
0.2 M
NaCI, at pH 7.4 and the suspension was dialyzed against a acetic acid (1:1000
or
0.5M) for 72 hrs at 4°C. Then, the suspension was frozen at -
80°C and lyophilized.
CA 02560161 2006-09-28
27
[00104] Example 4: Characterization of recombinant human collagen.
[00105] The total amino acid composition, including the percentage of the
collagen content in proline, hydroxy-proline, lysine and hydroxy-lysine, and a
partial
amino acid sequencing of the final product may be performed. The material for
analysis may be cut enzymatically before being analyzed. Electron microscopy
analyses can reveal the length, the periodicity and the overall organization
of the
collagen fibers, and thermostability can be evaluated also (Fertala et al.,
1994). For
example, Figures 7 and 8 show results of microscopic analysis of collagen
produced
according to the method described herein.
[00106] The gycosylation of procollagen can be assessed by testing its
affinity
with lectins, such as Concanavalin A, that specifically binds glusose and
mannose
residues. The purity, the respective molecular weights and amounts of a-1 and
a-2
chains of the processed collagen can be analyzed on SDS-PAGE. The confirmation
of the nature of the collagen can be tested on Western blots, using antibodies
directed specifically against human type I collagen. For example, Figure 9
shows
results of SDS-PAGE analysis of collagen produced according to the method
described herein. Figure 10 shows SDS-PAGE analysis together with Concanavalin
A-based staining of collagen produced according to the method described
herein.
[00107] The capacity of the collagen to polymerise into a gel can be assessed
by solubilizing the collagen in acetic acid 1:1000 and bring the solution to
physiological pH (7.2-7.5).
[00108] Although the present invention has been described hereinabove by way
of specific embodiments thereof, it can be modified, without departing from
the spirit
and nature of the subject invention as defined in the appended claims.
CA 02560161 2006-09-28
28
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