AU780813B2 - Stable expression of triple helical proteins - Google Patents

Description

AUSTRALIA

Patents Act 1990 Commonwealth Scientific and Industrial Research Organisation COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Stable expression of triple helical proteins The following statement is a full description of this invention including the best method of performing it known to us:la STABLE EXPRESSION OF TRIPLE HELICAL PROTEINS Field of the Invention This invention relates to the production of hydroxylated triple helical proteins by recombinant DNA technology. In particular, the invention relates to a method for producing hydroxylated triple helical proteins in yeast host cells by introducing to a suitable yeast host cell, DNA sequences encoding the triple helical protein as well as prolyl 4-hydroxylase (P4H), in a manner wherein the introduced DNA sequences are replicated, stably retained and segregated by the yeast host cells.

Background of the Invention .The collagen family of proteins represents the most abundant protein in mammals, forming the major fibrous component of, for example, skin, bone, tendon, cartilage and blood vessels. Each collagen protein consists of three polypeptide chains (alpha chains) characterised by a (Gly-X-Y)n repeating sequence, which are folded into a triple helical protein conformation. Type I collagen (typically found in skin, tendon, bone and cornea) consists of two types of polypeptide chain termed al(I) and a2(I) [i.e.

al(I) 2 while other collagen types such as Type II [ul(II) 3 and Type III [al(III) 3 have three identical polypeptide chains. These collagen proteins spontaneously aggregate to form fibrils which are incorporated into the extracellular matrix where, in mature tissue, they have a structural role and, in developing tissue, they have a directive role. The collagen fibrils, after 25 cross-linking, are highly insoluble and have great tensile strength.

The ability of collagen to form insoluble fibrils makes them attractive for numerous medical applications including bioimplant production, soft tissue augmentation and wound/burn dressing. To date, most collagens approved for these applications have been sourced from animal sources, primarily bovine. While such animal-sourced collagens have been successful, there is some concern that their use risks serious immunogenicity problems and transmission of infective diseases and spongiform encephalopathies bovine spongiform encephalopathy (BSE)).

Accordingly, there is significant interest in the development of methods of production of collagens or collagen fragments by recombinant DNA technology. Further, the use of recombinant DNA technology is desirable in that it allows for the potential production of synthetic collagens and collagen fragments which may include, for example, exogenous biologically active domains to provide additional protein function) and other useful characteristics improved biocompatability and stability).

The in vivo biosynthesis of collagen proteins is a complex process involving many post translational events. A key event is the hydroxylation by the enzyme prolyl 4-hydroxylase (P4H) of prolyl residues in the Y-position of the repeating (Gly-X-Y) sequences to 4-hydroxyproline. This hydroxylation has been found to be beneficial for nucleation of folding of triple helical proteins. For collagens, it is essential for stability at body ""temperature. Accordingly, the development of a commercially viable method for the production of recombinant collagen requires co-expression of P4H with the alpha chains. For mammalian host cells, co-expression of P4H will occur autonomously since these cells should naturally express P4H.

15 However, for yeast host cells, which for reasons of cost, ease and efficiency are more attractive for expression of recombinant eukaryotic proteins, transformation with DNA sequences encoding P4H will also be required.

Since P4H consists of a and p subunits of about 60 kDa and 60 kDa, yeast host cells for expression of recombinant collagen will require cotransformation with at least three exogenous DNA sequences encoding an alpha chain, P4H a subunit and P4H P subunit) and stability problems would therefore be expected if cloned on three separate vectors or, alternatively, all on episomal type vector. Indeed, even under continuous selection pressure, many episomal type vectors suffer stability problems if they are large or are present at relatively low copy number. An object of the present invention is therefore to provide a method for expressing triple helical proteins from yeast host cells wherein the introduced DNA sequences are stably retained and seeregated indenendnt of contin-nluou slcrtinn pressure.

Summary of the Invention Thus, in a first aspect, the present invention provides a method for producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: a first nucleotide sequence encoding prolyl 4-hydroxylase asubunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase p-subunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: wherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y in the triple helical forming repeating sequence (GlyXY) is proline, wherein A and D each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY) wherein B and C each represent a non-collagenous polypeptide or 25 peptide domain which does not comprise a triple helical forming repeating sequence (GlyXY)J, wherein n is an integer of from 2 to 1500, wherein 1 and p are each independently selected from 0 and 1. and wherein m and o are each independently selected from 0 and 1, with the proviso that at least one of m and o is 1, culturing the resulting yeast host cell of step under conditions suitable to express said P4Ha, said P4Hp and said polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein, wherein during culturing in step each of said first nucleotide sequence molecule, said second nucleotide sequence and said productencoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

In a second aspect, the present invention provides a yeast host cell which produces a hydroxylated triple helical protein upon culturing, wherein said yeast host cell comprises: a first nucleotide sequence encoding prolyl 4-hydroxylase asubunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, a second nucleotide sequence encoding prolyl 4-hydroxylase Psubunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is 15 one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: S" wherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y in the triple helical forming repeating sequence (GlyXY) is proline, wherein A and D each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY) wherein B and C each represent a non-collagenous polypeptide or peptide domain which does not comprise a triple helical forming repeating sequence (GlyXY),, wherein n is an integer of from 2 to 1500, wherein 1 and p are each independently selected from 0 and 1, and wherein m and o are each independently selected from 0 and 1, with the proviso that at least one of m and o is 1, wherein upon culturing of said yeast host cell, each of said first nucleotide sequence, said second nucleotide sequence and said product encoding nucleotide sequence(s) are replicated, stably retained and segregated by said yeast host cell.

In a third aspect, the present invention provides a triple helical protein produced in accordance with the method of the first aspect.

In a fourth aspect, the present invention provides a biomaterial or therapeutic product comprising a triple helical protein produced in accordance with the method of the first aspect.

In a fifth aspect, the present invention provides a method of producing, e psin yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: a first nucleotide sequence encoding prolyl 4-hydroxylase asubunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, S"(ii) a second nucleotide sequence encoding prolyl 4-hydroxylase SP-subunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula:

(A)

1 [Zi (CL wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY), wherein; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical i. forming repeating sequence (GlyXY),, and 1 and p are each independently selected from 0 and 1, B and C, which may be the same or different, each represent a 15 polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY), and m and o are each independently selected from 0 and 1; and culturing the resulting yeast host cell of step under conditions suitable to express said P4Ha and P4H3 and said synthetic polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein; wherein during culturing in step each of said first nucleotide sequence, said second nucleotide sequence and said product-encoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

O VO 25 In a sixth aspect, the present invention provides a yeast host cell capable of producing a hydroxylated triple helical protein upon culturing, said yeast host cell including: a first nucleotide sequence encoding prolyl 4-hydroxylase asubunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase P-subunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (D)p, wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY) (F)r] wherein; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and 15 i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY),, and 1 and p are each independently selected from 0 and 1, 25 B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY),, and m and o are each independently selected from n and 1; and wherein upon cilturing of said yeast host cell, each of said first nucleotide sequence, said second nucleotide sequence and said productencoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

In a seventh aspect, the present invention provides an hydroxylated triple helical protein comprising a polypeptide or peptide which is a synthetic polypeptide or peptide represented by the following formula: (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i wherein; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and i •X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, 15 A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, and I and p are each independently selected from 0 and 1, B and C, which may be the same or different, each represent a polypeptide or 00. peptide domain which is heterologous to collagen proteins and which does not 000° 20 comprise a triple helical forming repeating sequence (GlyXY),, and m and o are each 00 independently selected from 0 and 1.

In an eighth aspect, the present invention provides a method of producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: I t lll IIUL.lCIULlUC seqUC cllc olVUd g polUlrl -ll-lu Jiu u.-ouvmrJ.u (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase Psubunit (P4HP) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said m:\specifications\500000\500000\500223clmljc.doc polypeptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (A)I-(B)m-(GlyXY)n-(C)o-(D)p, wherein GlyXY represents a triple helical forming repeating sequence wherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y in the triple helical forming repeating sequence (GlyXY)n is proline, wherein A and D each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, wherein B and C each represent a non-collagenous polypeptide or peptide domain which does not comprise a triple helical forming repeating sequence (GlyXY)n, 15 wherein n is an integer of from 2 to 1500, wherein each of 1, m, o and p are selected from 0 and 1, with the proviso that at least one of m and o is 1, and when m is 1 and o is 0, 1 must be 1, and o is 1 and m is 0, then p must be 1, culturing the resulting yeast host cell of step under conditions suitable S 20 to express said P4Ha, said P4H3 and said polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein, 0 wherein during culturing in step each of said first nucleotide sequence molecule, said second nucleotide sequence and said product-encoding 0 nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

in a ocntr n aspfct, ep n n n prirnTcd a y esct horst cPl1 whinCih nmrdllPc a hydroxylated triple helical protein upon culturing, wherein said yeast host cell comprises: a first nucleotide sequence encoding prolyl 4-hydroxylase asubunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, a second nucleotide sequence encoding prolyl 4-hydroxylase Psubunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and m:\specifications\500000\500000\500223clmmjc.doc one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: wherein GlyXY represents a triple helical forming repeating sequence, wherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y in i: •the triple helical forming repeating sequence (GlyXY)n is proline, wherein A and D each represent a polypeptide or peptide domain which 15 optionally comprises a triple helical forming repeating sequence (GlyXY)n, wherein B and C each represent a non-collagenous polypeptide or peptide domain which does not comprise a triple helical forming repeating sequence (GlyXY)n, wherein n is an integer of from 2 to 1500, 20 wherein each of 1, m, o and p are selected from 0 and 1, with the proviso that at least one ofm and o is 1, and when m is 1 and o is 0, 1 must be 1, and when o is 1 and m is 0, then p must be 1, andl and p are each independently selected from 0 and 1, and wherein m and o are each independently selected from 0 and 1, with the proviso that at least one ofm and o is 1, wherein upon culturing of said yeast host cell, each of said first nucleotide j u,-i 4 1 A o -Ac c Vmr'Alrr t ,nr cTn o nucleotide sequence(s) are replicated, stably retained and segregated by said yeast host cell.

In a tenth aspect, the present invention provides a method of producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: a first nucleotide sequence encoding prolyl 4-hydroxylase a-subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, m:\specifications\500000\500000\500223clmmjc.doc (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase 3subunit (P4HP) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (B)m (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i wherein; 15 wherein GlyXY represents a triple helical forming repeating sequence; •i E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and ~i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, 20 Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or A A. Yf Vt.JIA LAJ J-AVlAjJk3J t tflI'Ak, .Ak t'LJ JJ1111, 11 6 %IatLA* sequence (GlyXY)n; B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n wherein each of m and o is 1 and each of 1 and p are selected from 0 and 1; and culturing the resulting yeast host cell of step under conditions suitable to express said P4Ha, P4Hp and said synthetic polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein; and m:\specifications\500000\500000\500223cmmjc.doc wherein during culturing in step each of said first nucleotide sequence, said second nucleotide sequence and said product-encoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

In an eleventh aspect, the present provides a yeast host cell capable of producing a hydroxylated triple helical protein upon culturing, said yeast host cell including: a first nucleotide sequence encoding prolyl 4-hydroxylase a-subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase 0subunit (P4HP) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when 15 hydroxylated, forms said hydroxylated triple helical protein, and wherein said S'i polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (B)m (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i wherein; wherein GlyXY represents a triple helical forming repeating sequence; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each inrl -nPnAPnt1r clotorl f-rcm i onr 1 anrl i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, m:\specifications\500000\500000\500223clmmjc.doc 3j A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n,, B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n, wherein each ofm and o is 1 and each of 1 and p are selected from 0 and 1, and; wherein upon culturing of said yeast host cell, each of said first nucleotide sequence, said second nucleotide sequence and said product-encoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

In a twelfth aspect, the present invention provides an hydroxylated triple helical protein i: comprising a polypeptide or peptide which is a synthetic polypeptide or peptide *i*i 15 represented by the following formula: (A)I (B)m (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i 20 wherein; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, wherein GlyXY represents a triple helical forming repeating sequence (~irr onrcPr ntr c t ri-I ao not-- X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, m:\specifications\500000\500000\500223cmmjc.doc B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n, wherein each of m and o is 1 and each of 1 and p are selected from 0 and 1.

In a twelfth aspect, the present invention provides an hydroxylated triple helical protein comprising a polypeptide or peptide which is a synthetic polypeptide or peptide represented by the following formula: (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i i wherein; i .E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, wherein GlyXY represents a triple helical forming repeating sequence, .Gly represents glycine, and S 20 X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from A •GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence

,VV\

B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n, wherein each of m and o is 1 and each of 1 and p are selected from 0 and 1.

Finally, in a thirteenth aspect, the present invention provides a biomaterial or therapeutic product comprising a triple helical protein according to the seventh aspect.

m:\specifications\500000\500000\500223clmmjc.doc Detailed disclosure of the Invention: The method according to the invention requires that the first and second nucleotide sequences encoding the P4H a and 3 subunits and the product-encoding nucleotide sequences be introduced to a suitable yeast host cell in a manner such that they are borne on one or more DNA molecules that are stably retained and segregated by the yeast host cell during culturing. In this way, all daughter cells will include the first, second and productencoding nucleotide sequences and thus stable and efficient expression of a hydroxylated triple helical protein product can be ensured throughout the culturing step and without the use of continuous selection pressure.

The method according to the invention can be achieved by; (i) integrating by homologous recombination) one or more of the exogenous nucleotide sequences one or more of the first, second and productencoding nucleotide sequences) into one or more chromosome(s) of the yeast host cell, or (ii) including one or more of the exogenous nucleotide sequences within one or more vector(s) including a centromere (CEN) sequence(s).

Alternatively, a combination of these techniques may be used or one or both of these techniques may be used in combination with the use of one or two high copy number plasmid(s) which include the remainder of the exogenous nucleotide sequences. For example, the first and second nucleotide sequences encoding the P4H x and 3 subunits may be integrated into a host chromosome while the product-encoding sequences may be included on vector(s) including a CEN sequence or on a high copy number vector(s).

~Preferably, the method of the invention is achieved by including the 25 exogenous nucleotide sequences within a vector(s) including a CEN sequence. Particularly preferred are the CEN sequence-including YAC (yeast artificial chromosome) vectors (Cohen et al., 1993) and pYEUra3 vectors (Clontech, Cat. No 6195-1). Other vectors including a CEN sequence may bhp generated by cloning a CEN sequence into any suitable expression vector.

Where one or more of the exogenous nucleotide sequences are included in a high copy number vector(s), it is preferred that the high copy number vector(s) is/are selected from those that may be present at 20 to 500 (preferably, 400 to 500) copies per host cell. Particularly preferred high copy number vectors are the YEp vectors.

The method according to the invention enables the production of hydroxylated triple helical proteins. The term "triple helical protein" is to be understood as referring to a homo or heterotrimeric protein consisting of a polypeptide(s) or peptide(s) which include at least a region having the general peptide formula: (Gly X in which Gly is glycine, X and Y represent the same or different amino acids (the identities of which may vary from Gly X Y triplet to Gly X Y triplet) but wherein X and Y are frequently proline which in the case of Y becomes, after modification, hydroxyproline (Hyp), and n is in the range of 2 to 1500 (preferably 10 to 350), which region forms, together with the same or similar regions of two other polypeptides or peptides, a triple helical protein conformation. The term therefore encompasses natural and synthetic collagens, natural and synthetic collagen fragments, and natural and synthetic collagen-like proteins (e.g macrophage scavenger receptor and lung-surfactant proteins) and as such includes any procollagen and collagen Types I-XIX) with or without propeptides, globular domains and/or intervening non-collagenous sequences and, further, with or 15 without native or variant amino acid sequences from human or other species.

Synthetic collagen and fragments encompassed by the term "triple helical protein" may also include non-collagenous, non-triple helical domains at the amino and/or carboxy terminal ends or elsewhere.

In accordance with the present invention, product-encoding nucleotide sequences are those which encode synthetic collagens and fragments thereof, and particularly those which encode a polypeptide(s) or peptide(s) of the general formula: (A) 1 -(B)m-(Gly X in which Gly is glycine, X and Y represent the same or different amino acids, the identities of which may i: vary from Gly X Y triplet to Gly X Y triplet but wherein Y must be one •25 proline, A and D are polypeptide or peptide domains which may or may not include triple helical forming (Gly X repeating sequences, B and C are intervening non-collagenous sequences which do not contain triple helical forming (Glv X V) repnRating suen.snr n is in the rano o f 2 to 1500 L -o (preferably, 10 to 300) and 1, m, o and p are each independently selected from 0 and 1.

In a variation of the present invention, the product-encoding nucleotide sequence(s) encode a synthetic polypeptide(s) or peptide(s) represented by the following formula: wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i (F)J.

wherein; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein 15 at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, and 1 and p are each independently selected from 0 and 1, B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n, and m and o are each independently selected from 0 and 1.

Preferably, in domain Z, the component (GlyXY)i has an amino acid 25 length which is at least three times greater than the combined amino acid length of the components E and F. Of course, in accordance with the formula of Z given above, one or both of E and F may be absent.

The portion of the product-encoding nucleotide sequence(s) encoding the repeat unit of domain Z may be generated through the use of polymerase chain reaction (PCR) techniques or chemical DNA synthesis. Stepwise addition of such nucleotide sequences, so as to generate the repeating nucleotide sequence for domain Z may be achieved by utilising different restriction sites at the termini of primers used to produce the PCR fragments or through variations in chemical DNA synthesis. The selected restriction sequences are such that the desired linear order of the repeated nucleotide sequences is achieved in a manner which maintains the overall phase or open reading frame of the product-encoding nucleotide sequence and which ensures that every third amino acid of the encoded Z domain is Gly. Example 7 hereinafter provides an example of this strategy. That is, Example 7 describes a strategy for producing a product-encoding nucleotide sequence encoding a domain Z with three repeat sequences derived from an integrin binding site of Type III collagen, wherein the first step was to clone an EcoRI- PCR fragment, the second step added a [(GlyXY),,-BssHII fragment, and the third step added a BssHII-[(GlyXY)nJ- SacII, wherein the amino acid sequence of the polypeptide encoded by each fragment is the same. This strategy can be readily extended to add a nucleotide sequence encoding fourth, fifth etc. repeat units.

i The product-encoding nucleotide sequence(s) may include a sequence(s) encoding a secretion signal so that the polypeptide(s) or peptide(s) expressed from the product-encoding nucleotide sequence(s) are 15 secreted.

Expression of the product-encoding nucleotide sequence(s) may be driven by constitutive yeast promoter sequences (e.g ADH1 (Hitzeman et al, 1981; Pihlajaniemi et al., 1987), HIS3 (Mahadevan Struhl,1990), 786 (no author given, 1996 Innovations 5, 15) and PGK1 (Tuite et al, 1982), but more preferably, by inducible yeast promoter sequences such as GALl-10 (Goff et al 1984), GAL7 (St. John Davis, 1981), ADH2 (Thukral et al, 1991) and CUP1 (Macreadie et al, 1989).

The first and second nucleotide sequences encoding the P4H a and P subunits can be of any animal origin although they are preferably of avian or 25 mammalian, particularly human, origin (Helaakoski et al., 1989). It is also envisaged that the first and second nucleotide sequences may originate from different species. In addition, the second nucleotide sequence encoding the P4H P subunit may include a sequence encoding an endoplasmic reticulum (ER) retention signal HDEL, KDEL or KEEL) with or without other target signals so as to allow expression of the P4H in the ER, cytoplasm or a target organelle or, alternatively, so as to be secreted.

Expression of the first and second nucleotide sequences may be driven by constitutive or inducible yeast promoter sequences such as those mentioned above. It is believed, however, that it is advantageous to achieve expression of the a and P subunits in a co-ordinated manner using same or different promoter sequences with same induction characteristics, but 6b preferably by the use of a bidirectional promoter sequence. Accordingly, it is preferred that the first and second nucleotide sequences be expressed by the yeast GALl-10 bidirectional promoter sequence, although other bidirectional promoter sequences would also be suitable.

Multiple copies of the first, second and/or product-encoding nucleotide sequences may be introduced to the yeast host cell present on a YAC vector or integrated into a host chromosone). It may be .o particularly advantageous to provide the product-encoding nucleotide sequence(s) in multicopy and, accordingly, it may be preferred to introduce the product-encoding nucleotide sequence(s) on a high copy number plasmid a YEp plasmid).

The introduced first, second and product-encoding nucleotide sequences may be borne on one or more stably retained and segregated DNA molecules. Where borne on more than one DNA molecule, the DNA molecules may be a combination of host chromosome(s) and/or CEN sequence-including vector(s) in combination with high copy number vector(s). Some specific examples of yeast host cells suitable for use in the method according to the invention, are transformed with the following DNA molecules: 1. YEp-P3 pYEUra3-cp, 2. YEp-P3 pYAC ca 15 3. YEpCEN-P3 pYEUra3-ap 4. YEpCEN-P3 pYACap pYAC-P3 pYAC xa 6. pYAC-P3 pYEUra3-ap pYACap-P3; wherein P3 represents a product-encoding nucleotide sequence(s), a and p represent, respectively, nucleotide sequences encoding the P4H a subunit and P4H P subunit, CEN represents an introduced centromere sequence. The pYEUra3 and pYAC vectors include CEN sequences.

Triple helical protein products produced in accordance with the 25 method of the invention may be purified from the yeast host cell culture by techniques including standard chromatographic and precipitation techniques (Miller Rhodes, 1982). For collagens, pepsin treatment and NaC1 precipitation at acid and neutral pH may be used (Trelstad. 1982).

Immunoaffinity chromatography can be used for constructs that contain appropriate recognition sequences, such as the Flag sequence which is recognised by an M1 or M2 monoclonal antibody, or a triple helical epitope, such as that recognised by the antibody 2G8/B1 (Glattauer et al., 1997).

Yeast host cells suitable for use in the method according to the invention may be selected from genus including, but not limited to, Saccharomyces, Kluveromyces, Schizosaccharomyces, Yarrowia and Pichia.

Particularly preferred yeast host cells may be selected from S. cerevisiae, K lactis, S. pombe, Y. lipolytica and P. pastoris.

As indicated above, it is particularly preferred that the first, second and product-encoding nucleotide sequences be introduced to the yeast host cell by transformation with one or more YAC vectors. YAC vectors are linear DNA vectors which include yeast CEN sequences, at least one autonomous replication signal ars) usually derived from yeast, and telomere ends (again, usually derived from yeast). They also generally include a yeast selectable marker such as URA3, TRP1, LEU2, or HIS3, and in some cases, an ochre suppressor sup4-o) which allows for red/white selection in adenine requiring strains the mutation of the adenine gene being due to i a premature ochre stop codon). More commonly, two yeast selectable markers are included, one on each arm of the artificial chromosome (each arm separated by the CEN). This allows selection of only those transformed 15 hosts containing YACs with introduced sequences of interest within the desired restriction cloning site. That is, correct insertion of the sequences of interest an expression cassette) rejoins the two arms of the restricted YAC, thus rendering transformants prototrophic for both markers. YACs have been designed to allow for the introduction of large exogenous 20 nucleotide sequences of the order of 100kb or more) into yeast host cells.

The present inventors have hereinafter shown that such YACs may be used for the stable expression of multiple exogenous nucleotide sequences (e.g.

nucleotide sequences encoding a natural collagen and both the a and P subunits of P4H).

In some embodiments of the invention, it may be preferred that one or more (but not all) of the first, second and product-encoding nucleotide sequences be introduced to the yeast host cell by transformation with one or two YED vectors. YED vectors carry all or oart of the yeast 2u plasmid with at least the ori of replication. They also include a yeast selectable marker such as HIS3, LEU2, TRP1, URA3, CUP1 or G418 resistance, and often also contain a separate ori, generally ColE1, and markers, such as ampicillin resistance, for manipulation in E.coli. They show high copy number, for example 20-400 per cell, and are generally efficiently segregated. Stability during cell division is dependent on the vector also containing the REP2/STB locus from the 2pi plasmid. However, stability is not as good as endogenous 2ut plasmid 9 of the host, particularly when heterologous genes are induced for expression. Stability also declines with increasing plasmid size. (Wiseman, 1991).

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components or features with or without the inclusion of a further component or feature or group of components or features.

The invention will now be described by way of reference to the following nonlimiting examples and accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES FIG. 1 shows, diagrammatically, the construction of the expression vector pYEUra3.2.12p#39a#5 (labeled pYEUra3-Mpa).

FIG. 2 shows the nucleotide sequence for the COLIII1.6 kb (DNA SEQ ID NO:39).

FIG. 3 shows, diagrammatically, regions of the human collagen III gene that have been 15 isolated by PCR. The 1.6 kb DNA used in the examples hereinafter is also shown. It is to be understood that the other regions shown in the figure could substitute for the COLIII1.6 kb DNA in those examples.

FIG. 4 shows, diagrammatically, the construction of the expression vector YEpFlagCOLIIII.6 kb (labeled YEpFlag-C3).

FIG. 5 shows, diagrammatically, the construction of pYAC5 .beta..alpha..

FIG. 6 shows, diagrammatically, the construction of pYAC .beta..alpha.-COL I111.6 kb.

FIG. 7 outlines the construction of synthetic collagen products.

FIG. 8 provides the nucleotide sequence (SEQ ID NO:40) for SYN-C3 together with 25 the amino acid sequence (SEQ ID NO:41) of the encoded polypeptide.

FIG. 9 shows single repeat cloned into EcoRI-Xmal Flag-EcoRI-XmaI-Flag-COOH

[FEX].

ICG. 10 shows two crontig1nlo reneat~ FlaP-EconRI-XmaI-BamHI-Fla-COOH

[FEXB]

FIG. 11 shows two repeat units with interruption Flag-EcoRI-XmaI-Flag-BamHI-SacII

[FEXS].

FIG. 12 shows Flag-EconRI-XmaI-BamHI-SacII-[FEXBS] DNA and Amino Acid Sequence of the YEpFLAG-hC3-PBD construct.

FIG. 13 shows Immunoblot of FLAG-tagged triple helical protein constructs with M2 anti-FLAG antibody following SDS-PAGE and Western transfer to nitrocellulose.

m:\specifications\500000\500000\500223clmmjc.doc 9a

EXAMPLES

Example 1 Construction of a Yeast Vector for Co-ordinated Co-expression of the a and (3 Subunits of Prolyl 4-hydroxylase Production of Yeast Expression Vector pYEUra3 (Clontech) contains the bidirectional promoter for GALl-10 expression.

Induction by galactose in the absence of glucose results in high level expression from pGAL 1 of any protein encoded by DNA sequences inserted in the correct orientation in the MCS (multiple cloning site) [either m:\specifications\500000\500000\500223clmmjc.doc Xhol, Sall, XbaI or BamHI sites] provided there is an initiating ATG start codon. For pGAL10, expression induced by galactose occurs if the DNA sequences to be expressed are inserted in frame with the ATG codon of when said DNA sequences to be expressed is inserted in the EcoRI site.

In order to utilise the EcoRI site for cloning, without the necessity that the insert be in frame with the ATG of GAL10 for expression, it was necessary to modify pYEUra3 to remove the GAL10 initiation codon. This was done as follows. A PCR fragment was generated using pYEUra3 as template and primers 3465 [5'CTG.TAG.Agg.atc.cCCGGG.TAC.GGA.GC-3', where the nucleotides shown in lower case code for a BamHI site] and primer 1440 [5'TTA.TAT.Tga.att.cTC.AAA.AAT.TC-3' where the nucleotides shown in lower case specify an EcoRI restriction site]. Primer 1440 introduces an EcoRI site preceding the initiating ATG of GAL10 in pYEUra3. The PCR fragment was restricted with BamHI and EcoRI and cloned into pYEUra3 similarly digested with BamHI and EcoRI, replacing the BamHI-EcoRI fragment containing an ATG start codon with a BamHI-EcoRI fragment lacking this ATG, to generate plasmid pYEUra3.2.12. The EcoRI site can then .i be used as a cloning site for which an initiating codon must be provided by 20 the inserted DNA sequence as with the MCS at the other end of the promoter, thus placing it under control of the bidrectional pGAL1-10 promoter and S• rendering expression inducible by galactose as are DNA sequences inserted in the MCS at the other end of the promoter. Cloning DNA sequences in the MCS and in the EcoRI site allows for co-ordinate expression by the bidirectional promoter when induced by galactose.

Isolation of DNA molecules encoding the a and psubunits of P4H: The a subunit of P4H was PCR amplified from cDNA (Clontech Human Kidney Ouick CloneTM cDNA Cat.#7112-1) usine primers 1826 TGT.AAA. ATT.AAA.gga.tcc.CAA.AG.ATG.TGG.TAT-3', lower case encodes BamHI site, ATG initiating codon for a subunit] and 1452 GCCG.gga.tcc.TG. TCA.TTC.CAA.TGA.CAA.CGT-3', lowers case encodes BamHI site, TCA translation stop codon]. Two isoforms were obtained and cloned into the BamHI site of pBluescript II SK+ [Stratagene Cat.# 212205] as storage vector to give pSK+a.1 (form I) and pSK+a.2 (form II) There are no BamHI sites in the DNA encoding the a subunit. The signal sequence for secretion is present in the BamHI fragment of both forms.

The p subunit of P4H [also known as PDI/protein disulfide isomerase] [Pihlajaniemi et al., 1987] was PCR amplified from cDNA (Clontech Human Kidney Quick CloneTM cDNA Cat.#7112-1) using primer pairs 2280

AC.TGG.ACG.GAT.CCC.GAG.CGC.CCC.GCC.TGC.

TCC.GTG.TCC.GAC.ATG-3'] and 2261 -G.GTT.CTC.CTT.ggt.

gac.cTC.CCC.TT-3', where the nucleotides shown in lower case encode a BstEII site] for the amino terminal part of the 0 subunit and primer pairs 2260 [5'-GAA.GGG.GAg.gtc.acc.AAG.GAG.AAC-3', where the lower case nucleotides encode a BstEII site] and 1932 TTA.GAC.TTC.ATC.TTT.CAAC.AGC-3'J for the carboxy terminal part of the 03 subunit. The two PCR fragments for the 0 subunit were then ligated ***together following BstEII digestion, to produce a single fragment encoding *:the entire p subunit. This fragment was then amplified using the primers 2280 GTC.TCC.GAC.ATG-3', where ggatcc encodes a BarnmHI- site, and ATG is the initiating codon of the P-subunit] and primer 1932 cTA.TTA.GAC.TTC.ATC.TTT.CAC.AGC-3', where ggatcc encodes a BamHI site and TTA is the translation stop codon for the 0 subunit] and then cloned into the BamHI site of pBluescript SKII+ to generate the storage vector 20 pSK+P. Subsequently, the BamiHI fragment of pSK+-P was amplified by using primers 2698 CTG.CTG-3', where gaattc encodes an EcoR site and the ATG. is the initiating codon of the 0 subunit] and 2699 CAG.TTC.GTG.CAC.AGC.TTT-3', where gaattc encodes an EcoRI site, and TTA. TTA. provides two translation stop codons, and GTG. changes a lysine residue to a histidine residue to provide a native yeast ER retention signal, HDEL His.Asp.Glu.Leu) ather than a mammalian KDAEL ER retention signal. The resultant PCR fragment was then blunt end cloned into the Srfl site of pCRScript [Stratagene, Cat.# 211190] to generate pCRScriptP.

After retrieving the EcoRI fragment containing the P subunit from pCRScriptP by EcoRI digestion, the fragment was again cloned into the EcoRI site of pCRScript to generate pCRScriptPEcoRI#4.

Construction of yeast expression vector including fragment encoding the a and p subunit of P4H: The 0 subunit fragment was obtained as an EcoRI fragment from EcoRI digestion of pCRScriptpEcoRI#4. This EcoRI fragment was cloned into the EcoRI site of pYEUra3.2.12 to generate plasmid pYEUra3.2.12p#39. The a subunit fragment from pSK+a.1 was re-excised from pSKa.l by BamHI and cloned into the BamHI site of pYEUra3.2.12p#39 to give pYEUra3.2.12p#39a#5] (Figure The P subunit fragment is under control of pGAL10 and the a subunit fragment is under control of pGAL1. This is a bidirectional promoter and allows co-ordinated induced expression of both subunits of prolyl-4-hydroxylase. Both fragments provide a native ATG initiating codon for translation. The encoded P subunit has its own signal secretion signal and a HDEL endoplasmic retention (ER) sequence at the .carboxy terminus of the protein. While the encoded a subunit with its own signal sequence has no ER retention signal it should, nevertheless, be retained through its interaction with the P subunit.

Example 2: Co-ordinated co-expression of a collagen segment and prolyl-4- S- hydroxvlase (a and 1 subunit) and synthesis of hydroxylated collagen Type 20 III in yeast A 1.6 kbp recombinant collagen fragment was generated by PCR using primers 1989 [Forward primer 5'-gct.agc.aag.ctt GGA.GCT.CCA.

GGC.CCA.CTT.GGG.ATT.GCT.GGG-3'] and 1903 [Reverse primer tcg.cga.tct.aga.TTA.TAA.AAA.GCA.AAC.AGG.GCC.AAC.GTC.CAC. ACC-3'] homologous to a region of the collagen type III alpha I chain (COL3A1). The template for isolation of the fragment of type III collagen alpha 1 chain was prepared from Wizard purified DNA obtained from a cDNA library [HL1123n Lambda Max 1 Clontech Lot#1245, Human Kidney cDNA Library].

The actual size of the isolated 1.6 kbp fragment is 1635 bp, comprising 1611 bp of COL3A1 DNA flanked either side by 12bp derived from the primers. The 1611 bp of COL3A1 DNA corresponds to nucleotides #2713-4826 (i.e codon #905-1442) of the full-length coding sequence, thereby spanning a portion of the a-helix region, all of the C-telo-peptide, all of the C-pro-peptide and stop codon.*l The nucleotide sequence for the COL3A1 DNA is provided at Figure 2. The region covered by the COL3A1 DNA is shown at Figure 3. The 1.6kbp fragment has a NheI [GCTAGC] site and a HindIII [AAGCTT] site added at the 5'-end and a XbaI [TCTAGA] site and a NruI [TCGCGA] site added at the 3' end [where the 5' end is taken to be the forward direction of the reading frame, ie the amino terminal end of the derived coding sequence, and the 3' end is that derived from the reverse primer corresponding to the 3' end of the gene and carboxy end of the derived amino acid sequence]. This confers portability on the collagen fragment.

The 1.6kbp fragment was cloned into the Smal site of YEpFlagi [IBI Catalogue #13400] so that the coding sequence is fused in frame with the vector expressed Flag protein. This allows for in frame expression of the introduced collagen gene fragment as a fusion protein when grown on ethanol. The blunt end cloning was performed by ligation of the SmaI digested vector sequence [gel purified] and the 1.6kbp PCR fragment [gel 15 purified, non-phosphorylated] at 20"C, in the presence of SmaI, to prevent recircularisation of the vector alone and reduce the level of false positive transformants obtained. There are no SmaI, NheI, HindIII, XbaI or NruI sites in the fragment of collagen DNA used in the cloning.

Small scale mini-preparations [prepared using BiolOl columns and described methods for their use] of DNA from ampicillin resistant transformant colonies of E.coli were screened by restriction enzyme analysis.

cultures rather than 1 ml cultures were required to prepare an adequate level of DNA for analysis, as YEpFlag plasmids do not appear to be at a high copy number in E.coli.

The fusion protein was of the form yeast a factor signal sequence for direction to the ER and commitment to the yeast secretion pathway, yeast a factor propeptide with cleavage sites for kex 2-endopeptidase, resulting in removal of all a-factor amino acid residues and generation of a free Flagtagged amino terminal end, Flag peptide for detection and tagging of the fusion protein (8 amino acid residues), linker peptide (4 amino acid residues), collagen helix (255 amino acid residues), collagen C-telopeptide [C-tel] (25 amino acid residues) and C-propeptide [C-pro] (255 amino acid residues) (for aid in formation of triple helix). The expected Flag-tagged protein consists of 547 amino acid residues with a expected MW of Expression of the fusion protein in YEpFlagl is under the control of the ADH2 promoter which is repressed by glucose but active in the presence of ethanol [a by-product of glucose metabolism]. There are multiple copies of the vector in individual yeast transformants due to the presence of the yeast 2 micron origin of replication in the vector, which leads to elevated expression of the 1.6 kbp PCR collagen fragment when glucose repression is lifted by consumption of glucose during growth. One unique feature of this cloning scheme is that inserts of the 1.6kbp collagen fragment in the wrong orientation will not form fusion products as the terminal leucine residue preceding the stop codon is coded by the codon AAT. In reverse orientation this generates a stop codon TAA. The result of incorrect insertion is the addition of only a single leucine coding codon [the stop codon TAA in reverse is AAT] following the Flag sequence before the protein is terminated.

The amino acid sequence of the Flag-tagged fusion protein at the point of fusion is N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-[Flag -Ala-Ser-Lys- Leu-[linker]-Gly-Ala-Pro-Gly-Pro-Leu-Gly-Ile-Ala-[a-helix].

15 The YEpFlag collagen construct [hereinafter referred to as YEpFlag COLII1.6kb; Figure 4] was introduced into a tryptophan prototrophic yeast strain such as for example BJ3505 [a pep4::HIS3 prb-1.6R HIS3 lys2-208 trpl- -101 ura3-52 gal2 canl], BJ5462 a ura3-52 trpl leu2-1 his3-200 pep4::HIS3 prb-1.6R cani GAL], (YGSG) JHRY1-5Da [a his4-519 ura3-52 leu2-3 leu2-112 20 trpl pep4-3] or KRYD1[ BJ3505xBJ5462 diploid] by transformation using electroporation, lithium acetate or spheroplast regeneration. Tryptophan auxotroph transformants were obtained, grown to high cell density in selective media [lacking tryptophan] followed by transfer to YPHSM, YEPM or YEPD or YEPGal, YEPE as described in the protocol provided with the YEpFlag expression system [IBI catalogue #13400]. At 3-9 days following inoculation iml aliquot's of culture were made and pellets and supernatants separated by centrifugation at 13000rpm in a benchtop centrifuge. Total yeast pellets were resuspended in 100l1 of gel loading buffer [5xSDS] containing PMSF [0.002M], vortexed vigorously for 2 minutes, and boiled for 5 minutes.

W A r, m ulI pe Illt IuuIA supernaiants were retained to which 100l 5xSDS/0.002M PMSF was added, and treated as described for the pellets. For both pellets and supernatants 20.l aliquot's were assayed by Western blot analysis of SDS-PAGE yeast total protein or of supernatants [media] following transfer to nitrocellulose and prehybridisation of the filters in blotto. Western blotting was carried out using a-Flag MAb M1 [against N-terminal free Flag] (International Biotechnologies Inc., (Eastman Kodak) Cat. No. IB13001) or M2 [against Flag] (International Biotechnologies Inc., (Eastman Kodak) Cat. No.

IB13010).

Western blots revealed the presence of a protein band of approximately 60kDa. This is the expected size of a protein fusion containing Flag-helix-C-tel-C-pro. After prolonged incubation the Flag responsive antibodies detected the appearance of the fusion product in the media.

Detection in both pellet and media supernatant with Ml antibody demonstrates that the a factor leader has been completely removed. No precursor forms with ox factor pro-region [glycosylated or not] were observed.

No band corresponding to 60kDa was obtained which hybridised to M1 or M2 with proteins obtained from untransformed yeast hosts. When yeast transformed with YEpFlag [no insert] alone was used, bands were obtained in pellets, but only with M2 MAb. These bands correspond to unsecreted a-proregion-with C-terminal Flag and various glycosylated forms of 15 the same. No Flag is detected in supernatants but this is to be expected as it .is only 8 amino acids long. No expression from the ADH2 promoter for any construct is observed in the presence of glucose.

YEpFlagCOLIII 1.6kb was also co-introduced [co-transformed] into yeast strains such as BJ5462 and KRDY1 which are capable of growth on 20 galactose along with pYEUra3 [Clontech ][pYEUra3 and its derivatives *contain the bidirectional GALl-10 promoter. Both the ADH2 and promoters are repressed by glucose. The GALl-10 promoter is induced by galactosel] or pYEUra3.2.12 [a modification of the Clontech parent vector which allows cloning of genes into an EcoRI site without the necessity of the introduced gene being in the correct reading frame] or pYEUra3.2.12p#39 [in which the DNA encoding the P subunit (equivalent to protein disulfide isomerase of prolyl-4-hydroxylase is cloned into the EcoRI site of pYEUra3.2.12 under control of GAL10 promoter] or pYEUra3.2.12p#39a#5 [in which the DNA encoding the a subunit of P4H is cloned into the BamHI J _,rrT T .n T 3u oiLc ui pAjUralaO...L2p-Ot3#9 llutiI uunuui Ut Ie GALl promoterj.

Transformants were selected on media lacking tryptophan or uracil or lacking both tryptophan and uracil. As previously done with tryptophan transformants obtained above with YEpFlag or YEpFlagCOLIII1.6kb, transformants were grown in selective media prior to growth in YPHSM, YEPM, YEPD ,YEPG or YEPE and after 4 days galactose was added to a final concentration of 0.5% or Total yeast protein or supernatants were analysed by Western blot analysis as described above except that a third MAb [5B5 against the P subunit] (Dako Corporation, Cat. No. M877) was also used.

Western blot analysis revealed the presence of a -60kDa band in trp or trp- ura- yeast transformed with YEpFlag COLIII1.6kb but not YEpFlag alone when screened with MAb M1 or M2 as was previously the case with transformants obtained with single plasmid transformation.

Analysis also showed the presence of a -60kDa band in ura- or ura trp- but not trp- yeast transformants transformed with pYEUra3.2.12p#39 or pYEUra3.2.123#39a#5 or cotransformed with same plus YEpFlag or YEpFlagCOLIII 1.6kb when screened with anti-p subunit MAb 5B5 but only following induction with galactose and only when galactose was between 0.2 and 0.5% and not at The expected size for the 0 subunit is also This band is not detected by M1 or M2 in uracil auxotrophic yeast 15 transformed with pYEUra3.2.12P#39 or pYEUra3.2.12p#39a#5 alone.

At the time ofthe experimentation, an antibody for the detection of expression of the a subunit from the bidirectional GAL1,10 promoter in pYEUra3.2.12p#39a#5 was not available but as the promoters for both GALl and GAL10 are normally co-induced and under the control of the same UAS 20 (upstream activation sequence) in yeast it was assumed that the a subunit is also transcribed and expressed where the P subunit is demonstrated to be expressed. To test this, the capacity for pYEURa3.2.12p#39a#5 YEpFlag COLIII 1.6kb co-transformants induced with 0.2% galactose following at least 4 days growth on YPHSM to produce functional P4H was examined.

25 Galactose was added following the clear demonstration of the expression of Flag-collagen by a positive response of yeast protein to M1 or M2 in Western blots and the absence of a response to MAb 5B5 against 0 subunit. Following iniduction with oalactnos [l hr nrntp.in was again examined and the

A

presence of M1 or M2 responsive bands and 5B5 responsive bands were separately demonstrated. Protein was transferred to PVDF membrane following SDS-PAGE and the membrane sliced into strips. Membrane strips containing protein from the region corresponding to the 60kDa responsive area was subject to hydrolysis and amino acid analysis. Amino acid analysis revealed the presence of hydroxyproline in this material from cotransformants of yeast co-transformed with YEpFlagCOLIII1.6kb and pYEUra3.2.12p#39a#5 after induction with 0.2% galactose but no hydroxyproline was detected with protein from control samples with or without galactose.

The media used contains peptone derived from bovine protein hydrolysates but no hydroxyproline was found in total yeast grown on this media nor in any of the singly transformed yeast [one vector alone]. Only in yeast co-transformants was hydroxyproline detected in the 60kDa bands and then only when galactose was added. Uninduced co-transformants [no galactose] in which Flag detected collagen was expressed did not contain any hydroxyproline in the 60kDa band excised from PVDF following transfer.

Hydroxyproline was only found in the 60kDa region and not in other regions of the blot.

The clear evidence then, is that following galactose induction of pEUra3.2.123#39a#5 a product is produced in yeast which is capable of hydroxylating the proline residues of a co-expressed Flag-tagged collagen 15 fragment. Such activity is not found in yeast untransformed or transformed with pYEUra3.2.12p#39 [no a subunit] or in uninduced yeast grown on ethanol or glucose.

A clear advantage of this method of co-expression for the production of hydroxylated collagen in yeast is the co-ordinated expression of the three genes that is possible in co-transformants. Another advantage is that the a and p subunits themselves are co-ordinately expressed. A third advantage is that the ap expression vector pEUra3.2.12p#39a#5) contains a centromere sequence and behaves as a mini-chromosome. It is therefore very stable and does not require selection pressure to be maintained for its stability. The removal of selection pressure in yeast does not appear to effect the stability of the YEpFlag collagen construct as it is in very high copy number, but clearly the ability to only be concerned with maintenance of a single plasmid in -the asenrce nf pseletion nrepsiir' is imnnrtant rather than balancing the effects of selection pressure on the stability of three separate plasmids if the a, P and collagen fragments were separately cloned on multicopy vectors. Also the use of a bidirectional promoter to express the a and 0 subunits simultaneously is of benefit rather than expressing them from different promoters on different plasmids in different amounts. The a subunit probably requires the synthesis of equal or higher levels of the P subunit for its correct assembly into functional P4H (0 2 f 2 enzyme and coordinated expression appears to be an efficient mechanism to ensure this.

*11 I odon niumrieing for collagen type Ml alpha 1 chain: ATG. codon codoui #1-codon #24, signal sequence; iodon #25-(:odon #116, N-pio-peptide seiluenco. codoui #117-codon #130. N-telo-peptide seq te nce: codon #131codoji #1161. ax-helix sequence; codoui #1162-codon #1186, C-telo-peptide; codon #1187-codon #1441, C-pro-.

peptido: (:0(101 #1442, stopl and Jcnrresponding nucleotide numbering for collagen typo Ill alpha 1 chain: nu1cleotido #1-72. signnl sequence; nucleotide #73-348. N-pio-peptido sequence; nucleotide #349-390. N-telo-peptide; nuclootide #31) I-nt#3983. ax-helix regionu; nucleotido #3984-4058, C-telo-peptide; nucleotide #4059-4823, C-pro-peptide sequenice; uumclotjde #4824-4826, stop codon].

Example 3: Use of Yeast Artificial Chromosomes [YACs1 for co-ordinated expression of the a and 13 subunits of Prolyl-4-hydroxylase IP4H].

[11454bp] (Kuhn and Ludwig, 1994) was digested with BarnHIf to liberate the HIIS3 gene [l2l0bp] from between the 2 telomiere ends and with SalI-NruI to produce two fragments [left arm: fragment 1, 5448bp right arm: fragment2, 4238bp] which were gel purified. Fragment 1 was Bamff-ltelomere end-E. coli ori-f3-lactaniase gene [ampicillin-resistancel -TRP1-ARS 1- CEN4-RNAsup-o-SalI. Fragment 2 was Bai-l-teloinere end-URA3-NruI.

pYEUra3.2.12f3#39oc#5 was digested with SalI-EcoRV to produce a P4H expression cassette fragment of the form SalI-XbaI-BainHI-aX-ATG- BamH-pGALl1 i-EcoRI-ATG-O3-EcoRI-S mal-EcoRV [4864bp] which was gel purified. The expression cassette fragment encoding the ac and f3 subunits of P4H under the control of a galactose inducible bidirectional promoter was :2;:*ligated with fragments 1 and 2 of the BamI-I-SalI-NruI digested pYAC5 and the ligation mix used to transform the following yeast strains: BJ2407 a/ax prbl-11222/prbl-1122 prcl-407/prcl-407 pep4-3/pep4-3 leu2/leu2 trpl/trpl ~**ura3-521ura3-52 KRYD1 a/cc ura3-52/ura3-52 trpl-A1O1/trpl lys2-208/LYS2 HIS3/his3A200 gal2/GAL2 cani/cani pep4: :IS3/pep4: :L-S3 a prblAl.6R/prbAl.6R 1, GYl leu2 adel trpl ura3 1, JI-RY1-5Dcx [ac his4-519 ura3-52 leu2-3 leu2-112 trpl pep4-3 1, and YPH15O[ a/a ura3-52/ura3-52 lys2- 801a/lys2-801a adel-101o/adel-l0io leu2Al/leu2Al trpl-A63/trpl-A63 his3A200/his3A200 using the method for lithium acetate transformation.

Yeast strains were also transformed with pYAC5 digested with Bami-i and undigested Ura' Trp' co-trans forma nts were obtained for all strains where the two fragments of pYAC5 each carrying either TRPI [SalI-CEN4-TRP1-Bam~l] [fragment 1] or UFA3 [NruI-URA3-BamJiIf] [fragment 2] as the selectable marker for transformation each on one arm of the YAC, had been linked together by the insertion of the P4H expression cassette into the SalI-EcoRV sites. This vector was designated pYAC50(x (Figure The vector was of the form BamHI-telomere-URA3-NruI/EcoRV [both sites destroyed]-P-ATGpGAL10-1-ATG-a-SalI-tRNAsup-CEN4-ARS1-TRP1-AMPr-ori-telomere- BamHI. The presence of the CEN4 sequence means the vector behaves as a stable chromosome during replication and is segregated at least 1 copy per cell at mitosis and meiosis [as was the case for pYEUra3.2.12p#39a#5]. The telomere ends mean that the vector is linear and stable.

Transformants and controls [pYAC5 alone (circular), linearised by BamHI digestion] were replica plated onto nitrocellulose filters laid over selective media [SD Complete lacking uracil and tryptophan] or rich media [YEpD] and incubated 2-5 days at 30C till confluent. Filters were transferred to selective media containing galactose instead of glucose or rich media containing galactose as well as glucose media plates and grown at 30C for periods between 2h-72h. At the end of incubation colonies were lysed on 0.1%SDS-0.2N NaOH-0.1% P-mercaptoethanol, washed with 15 water and filters blocked with Blotto. Production of the a and 3 subunits of P4H was ascertained by hybridising the treated filters with MAbs specific for the a [MAb 9-47H10] (ICN Biomedical Inc. Cat. No. 631633) and P [MAb subunits. Colonies transformed with pYAC5poa and induced with galactose showed hybridisation with VIAbs against the subunits of P4H demonstrating co-ordinated production of a and P from the bi-directional GAL 1-10 promoter. Controls filters and control yeast did not produce a response to P4H MAbs. Yeast transformants carrying pYAC5pa grown on glucose [a repressor of the bi-directional GAL 1-10 promoter] also did not produce a positive response.

Positive transformants identified in the above screening procedure were precultured/grown in 10ml liquid culture media containing selective media lacking ura and trp or rich media [containing glucose, glycerol or rnffinnsp1 Alinllnt s w rp transfp.rrp.d to indilring media [.sPlective or richl -1 -j containing 0.2-2% galactose. Where glucose was the carbon source pellets were washed in sterile water prior to induction. After 2-20h further growth at 30C cell pellets were collected, suspended in loading buffer and total yeast protein separated on SDS-PAGE and western blotted. Filters were blocked with blotto and hybridised with MVAbs against both of the P4H subunits.

Only those yeast transformants carrying pYAC53a and induced with galactose gave the expected 60kDa bands for a and P subunits. This demonstrates that the P4H expression cassette has been functionally inserted into pYAC5. The advantage of having the P4H cassette in the pYAC is twofold; as with the case of pYEUra3.2.12p#39a#5 the presence of the CEN sequence means that the vector is stably maintained in this system when selection pressure is removed for growth in rich media, which increases yield through increased cell density, and the pYAC5 pa construct allows for the subsequent insertion of multiple and different triple helical protein expression cassettes.

Example 4: Co-expression of collagen/triple helical protein fragment(s expressed on a multicopy plasmid and P4H subunits in yeast transformants carrying pYACSp3a.

Yeast host strains containing pYAC53a or pYAC5 were transformed with YEpFlagColIII 1.6kb or YEpFlag alone. The form of the collagen bearing vector was circular and multicopy. In this instance, as the YEpFlagCOLIII :i 15 1.6kb and the pYAC constructs both contain the same selectable marker, yeast transformants producing Flag tagged-collagen were identified by colony hybridsation with MAbs against Flag [Ml or M2]. Colonies were also .i screened for whether they carried extra copies of bla gene [multicopy] by identifying those colonies producing increased levels of p-lactamase by PADAC assay (Macreadie et al., 1994). In other examples, the multicopy •plasmid could utilise a different selectable marker other than URA3 or TRP1 found on each arm of the YAC. Various co-transformant types carrying pYAC53cpa and YEpFlag COLIII 1.6kb were assayed as in Example 1 for collagen production, P4H subunit production, and P4H activity. Those co- 25 transformants containing pYAC53p plus YEpFlag COLIII 1.6kb were then screened as described in the previous example for hydroxylated collagen to identify 60kDa bands in western blots responding to MAbs against the a and p and Flag following induction. The a and 3 subunits were only identified following galactose induction. Hydroxylated protein was only identifed following induction of both the a and P subunits of P4H.

Example 5: Introduction of collagen expression cassette into pYAC5 and YEpFlag was linearised by digestion with Scal which cuts at a single recognition site in the ampicillin resistance gene for p-lactamase [bla]. There are no Scal sites in the 1.6kb collagen fragment insert so Scal could also be used to linearise YEpFlagColIII 1.6kb. Linear DNA was used to transform yeast containing pYAC5 or pYAC5pa. Yeast transformants producing Flag tagged-collagen were identified by colony hybridsation with MAbs against Flag [Ml or M2]. Colonies carrying extra copies of bla gene [multicopy] were also identified. Those colonies producing increased levels of p-lactamase by i the PEDAC assay were found to have inserted a copy of YEpFlag COLIII 1.6kb i into the pYAC5 or pYAC53a vector of the host strain and correspond to those colonies positive to MAbs M1 or M2. The increased P-lactamase activity is a result of gene amplification resulting from homologous recombination between the linearised bla gene on YEpFlagCOLIII 1.6kb and the bla gene on pYAC. The new plasmids formed by insertion into pYAC5 or pYAC5pa of the YEpFlag COLIII 1.6kb vector were designated pYAC-COLIII 1.6kb and pYAC ca-COLIII 1.6kb (Figure Expression experiments were performed and only those strains carrying all 3 genes on the YAC [pYAC pa -COLIII 1.6kb] and induced for P4H with galactose produced hydroxylated collagen.

o Example 6: Cloning and expression of a synthetic collagen protein.

A strategy is described for the generation of "synthetic/novel" collagen proteins involving the in vitro assembly of synthetic 25 oligonucleotides repeat sequences encoding the peptide GPP.GPP.GXY (where XY LA, ER, PA or AP). The synthetic collagen sequences are engineered to contain a high percentage of proline residues as this residue has been shown to confer thermal stability to collagen molecules. The residue pairs chosen for the XY position LA, ER, PA or AP), are selected since they appear in statistically higher amounts in fibrillar collagens.

Mixtures of synthetic oligonucleotides encoding GPP.GPP.GXY may be joined together to generate DNA fragments of discrete lengths, encoding synthetic collagen proteins of discrete molecular size and with different physical characteristics. These synthetic gene segments can be cloned into various expression vectors for subsequent production of a collagen product in yeast. An outline of the strategy for construction of a synthetic oligonucleotide encoding a collagen is shown in Figure 7 where XY is shown, for the purposes of exemplification only, as ER, LA, AP, PA.

Such synthetic oligonucleotides have been synthesised and several libraries containing gene segments of various lengths have been generated by ligating these oligonucleotides together (maximum visible DNA length approx. 1000 base pairs coding for a polypeptide of 350 amino acid residues).

Example 7: Construction of a synthetic hydroxylated triple helical protein for stable expression in yeast.

A region of Type III collagen was selected for its known capacity to bind and activate platelets [through an integrin binding site near -Gly-Leu- Ala-Gly-Ala-Pro-Gly-Leu-Arg]. A region of 5 GLY-X-Y repeats to the Nterminal side and 7 GLY-X-Y repeats to the C-terminal side were also 15 included to form the basic repeat unit for inclusion in the synthetic fragment.

The sequence of the repeat was GGKGDAGAPGERGPP-GLAGAPGLR- GGAGPPGPEGGKGAAGPPGPP. This corresponds to residues 637-681 (nucleotides 1909-2043) in the COL3A1 gene [with Met At the 5'-end of the DNA an EcoRI site and NheI site was included such that the NheI site provided an initiating methionine. Thus the sequence at the amino end is •MGAPGAP, where GAPGAP is the natural sequence flanking the repeat in COL3A1. The repeat was linked to a second repeat by a linker which introduced a Bspl20I site for later manipulations and provided the sequence SGGP between the first and second repeat unit. The second repeat was linked 25 to a third repeat by a linker which introduced a BssHII site [again for later manipulation] and resulted in the amino acid sequence GAR. The third repeat was flanked by 2 additional GPP triplets, a GCC triplet and finally GLEGPRG. This was a result nf inclurlino crnrinp se.niiqncr that nrovided for XhoI, SacII and Nhel sites. These were included for flexibility of cloning at later stages. The NheI site provides an in frame stop codon.

The synthetic fragment was produced by PCR from primers against COL3A1 in 3 pieces initially. Fragment 1 was EcoRI-NheI-Met-[GAP]2- [REPEAT ]1-Bspl20I. The primers for this were ggtgctccaggtgctcc-3' [up] [primer U101] and 5'-ggcc-acctggtggacctggtgg-3' [down] [primer D101]. The second PCR fragment used primers ggtggtaagggtgacgc-3' [up] [primer U102] and 5'-cgcgc-acctggtggacctgg-3' [down] [primer D102]. For the 3rd repeat primer pairs used were ggtggtaagggtgacgctgg-3' [up] [primer U103] and tggtggacctgggtgg-3' [down] [primer D103]. The three fragments form the PCR reactions were gel purified and ligated together. The DNA from the ligation mixture was then used as the template for a further round of PCR using primer U101 and a new primer at the 3' end acaaccctggtgg-3' [down] [primer D104]. A band of approximately 500 bp was produced and gel purified, digested with EcoRI-Nhel and ligated to pYX141 (Ingenous Cat. No MBV-025-10) [LEU2-CEN-p786] also digested with EcoRI-NheI before being transformed into E.coli. Transformants were screened by PCR using primers for the second fragment and DNA from positive colonies were miniprepped and screened by enzyme digestion with S. EcoRI-NheI for the presence of an insert of approximate 500 bp. This storage vector was designated pYX-SYN-C3-1. The EcoRI-NheI fragment was transferred to pYX243 [2u-LEU2-pGAL] (Ingenous Cat. No IMBV-035-10) to give pYX-SYN-C3-2 and this plasmid was introduced into a yeast host cell including neucleotide sequence for the carrying the P4H a and 3 subunits [either pYEUra3.2.12p#39a#5 or pYACa3]. Expression following galactose induction was determined by using a MIAb 2G8/B1 (Werkmeister Ramshaw, 1991) which recognises the sequence GLAGAPGLR. An EcoRI-SacII fragment •from pYX-SYN-C3-2 was also introduced into the EcoRI-SacII of YEpFlag to produce YEpFlag-SYN-C3 and this too was introduced into a yeast host cell expressing P4H on induction by galactose. A product of approximately 18 kDa [the expected size of SYN-C3] was detected in yeast induced with 25 galactose by Western blotting.

The nucleotide sequence for SYN-C3 is provided at Figure 8 together with the amino acid sequence of the encoded product.

Example 8: The use of yeast other than Saccharomvces cerevisiae.

The GALl-10 promoter is functional in Kluyveromyces whilst the ADH2 promoter is constitutively expressed in S. pombe. By shifting the expression cassettes to appropriate vectors, other yeast hosts can be used.

K. lactis for instance has been shown in some instances to display less proteolytic activity for recombinant products. Alternatively, P. pastoris could be used for multiple integration of the expression cassette for a P into the chromosome.

For expression in P. pastoris, the nucleotide sequence described in the previous example encoding the synthetic triple helical protein [SYN-C3] was inserted into the P. pastoris vector pPIC9 (Invitrogen, Cat. No. K1710-01) at the EcoRI-NotI sites [pPIC-SYN-C3]. Following digestion with either BglII or Sail, the plasmid was introduced into P.pastoris where it was integrated at either the AOX1 or HIS4 sites for BglII or SalI respectively. The nucleotide sequences encoding the P4H a and 0 subunits were also introduced into P.

pastoris using the EcoRI site of pHIL-D2 (Invitrogen, Cat. No. K1710-01) for the p subunit and integration at HIS4 and the BamnHI site of pHIL-S1 (Invitrogen, Cat. No. K1710-01) for the a subunit and subsequent integration HIS4. All three expression cassettes were under the control of the AOX1 promoter and induced by methanol.

Example 9: Enhanced expression of proly-4-hydroxylase a and 1 subunits 15 from the GALl-10 promoter by use of yeast with different backgrounds for control of galactose induced expression.

The plasmid pYEUra3.2.12P#39a#5 [encoding the a and P subunits S- of P4H under the control of the GALl-10 bidirectional promoter] can be introduced into a yeast host cell with the following genotype a or a, ura3 trpl egdl bttl. In these cells, the absence of the products for the EGD1 and BTT1 genes results in higher levels of galactose induced expression from GAL4 dependent promoters such as GAL2, GAL4, GAL7, GALl-10, MEL1 (Hu Ronne, 1994).

Another mechanism for enhanced expression is the use of a yeast 25 host cell carrying multiple copies of the GAL4 (Johnston Hopper, 1982) positive transcriptional activator under its own controlled induction by galactose. This leads to enhanced expression as there is no limit to the availability of the transcriptional activator for the GALl-10 oromoter.

Similarly, the yeast host cell could contain multiple copies of the SGE1 gene (Amakasu et al., 1993) which also leads to enhanced transcription from galactose induced promoters.

Various combinations of these backgrounds could also be utilised; that is egdl bttl SGE1"" or egdl bttl GAL4"C or egdl bttl SGE1'"" GAL4 m [where mc represents multiple copies].

Example 10: Expression of collagen from promoters other than ADH2.

The collagen encoding nucleotide sequence in YEpFlag COL 1.6kb can be excised as a NheI or HindIII- XbaI or NruI fragment for insertion into other fusion vectors under the control of other promoters. Alternatively, the pADH2-a signal-A-proregion-Flag collagen cassette can be excised as a NaeI or SacI BglII or XbaI or Spel or SnaBI or NotI, for example, and introduced into an appropriate vector such as YEplacl81 (Gietz Sugino, 1988) or pMH158 (Heuterspreute et al., 1985) for expression in different copy numbers and host backgrounds or into vectors with CEN sequences. Alternatively, CEN sequences can be introduced into the YEpFlag vector itself. The cassette can also be removed without the ADH2 promoter using NruI and introduced into an appropriate vector behind an appropriate promoter.

Collagen encoding nucleotide sequences can be expressed using the CUP1 promoter in vectors such as pYELC5 (Macreadie et al., 1989) as an alternative to the ADH2 promoter. This promoter is induced by addition of copper copper sulfate) and may have the advantage of an increased reducing environment and enhancement of P4H activity during co- Sexpression. A second promoter that can be used is the TIP1 promoter which is induced by cold shock. Here the stability of the expressed collagen may be enhanced without the need for hydroxylation by inducing expression by shifting growing yeast from 30 0 C to 18 0

C.

The method according to the invention provides for the stable expression of triple helical proteins from yeast host cells. The products of the method may be natural and synthetic collagens, natural and synthetic collagen fragments and natural and synthetic collagen-like proteins.

Synthetic products may show enhanced or novel functions inclusion of RGD and/or YIGSR sequences from fibrorectin and laminin). The products may be u.ed in a w.'ide rang onf applictions inrluding binimn1lnt production, soft and hard tissue augmentation, wound/burn dressings, sphincter augmentation for urinary incontinence and gastric reflux, periodontal disease, vascular grafts, drug delivery systems, cell delivery systems for natural factors and as conduits in nerve regeneration.

Example 11 A DNA fragment encoding the mature long form of PDGF B lacking the secretion signal sequence), was amplified by PCR from clone pYEULC-BX/3E1 Sleigh, CSIRO, personal communication) which encodes the full length B form of PUGF (Martins, RN, Chelboun, JO, Sellers, P, Sleigh, M, and Muir, J. Growth Factors 1 299 306, 1994); cloned into the BamHI-EcoRI sites of pYEULC-BX, and under transcriptional control of an inducible yeast promoter (Macreadie and Vaughan Recent Res. Devel. in Biotech. Bioeng. 1, 465-479 1998) using the following pair of oligonucleotides: N8628 5'-GATCCGTCGACGAGGGGGACCCCATTCCCGAG-3' as the forward primer; and N8617 5'-GCGGCCGCTTACCGCGGGCTCCAAGGGTCACCTT-3' as the 15 reverse primer.

The PCR fragment generated in this way was gel purified and then subjected to restriction enzyme digestion with Sail and Sacll. Following further gel purification the PCR fragment was directionally cloned into the similarly digested vector pYEpFlag-EXB (ie. linearised with Sail and Sacll) to generate YEpFlag-EXB-PDGF1.

This results in a construct in which the gene sequence for the mature long form of PDGF-B is in the correct orientation with respect to the triple helical sequences which produces an in frame fusion of the PDGF-B sequence with the two in tandem copies of o: *the triple helical repeat unit preceding it in the vector at the carboxy terminus).

An additional protein construct using YEpFlag-EXB-PDGF1 as the base vector was used to generate YEpFlag-EXBS-PDGF-B which has 3 tandem triple helical repeat units preceding the in frame C-terminal fusion of PDGF-B. The third copy of the triple helical repeat unit xxwa opnerated .s n PCR fracment from template YEDFlag-EXBS using the oligonucleotide pair: N8618 5'-GGGCCCGGATCCGGTGGTAAGGGTGACGCTGGGTGC-3' forward primer; and N8619 5'-CGCGGGTCGACTGGTGGACCTGGTGGACCAGCAGC-3' as the reverse primer.

m:\specifications\500000\500000\500223clmmjc.doc The PCR fragment thus generated and encoding the triple helical repeat unit was digested with BamH1 Sail, re-purified on agarose gels and directionally cloned into the BamHl Sail sites of YepFlag-EXB-PDGF-B, (linearised with BamHI -Sall) between the two preceding triple helical repeat units and the PDGF-B sequence. The final form of the chimeric protein produced from YEpFlag-EXBS-PDGF-B is of the form NH 2 factor pre-pro]-[Flag] (triple helical repeat region [repeat 1-repeat 2-repeat 3)-PDGF-B- COOH. These two constructs encoding C-terminal PDGF fusions are transformed into yeast as described previously.

Example 12: Construction of a single repeat cassette unit for a synthetic hydroxylated triple helical protein for stable expression in yeast.

A region of type III collagen was selected for its known capacity to bind and activate platelets as described in Example 7. A single repeat unit was introduced into 15 the yeast expression vector YEpFlag between the EcoRI and XhoI, by addition of EcoRI-NcoI sites at the 5'-end of the basic repeat unit and a XmaI site at the 3'-end. The cloned first repeat is thus flanked at the NH 2 -end by the amino acid sequence NSGAM [arising from the EcoRI-NcoI linker], with the addition of the amino acid sequence GAPGAP from the collagen type III native sequence, and is terminated at the -COOH S. 20 end by addition of GGP [from the Xmal site] followed by the sequence GRSIDGSGPVDPA, resulting from the in-frame fusion with the YEpFlag cloning vector sequences (see Figure The amino acid sequence thus reads

NSGAM-GAPGAP-

GGKGDAGAPGERGPP-GLAGAPGLR-GAGPPGPEGGKGAAGPPGPP-GGP-GRS-

25 IDGSGPVDPA. This construct was designated pFEX. The basic repeat sequence (described in example 7) is shown in bold.

Yeast cells were transformed with the construct and grown as described in the Sigma manul Siinprn~ tnt was collected following centrifugation at 1000rpm to remove cells. The supemantant was then run on SDS-PAGE and transferred by Western blotting to nitrocellulose. The blot was hybridised against anti-FLAG MAb M2 followed by hybidisation with secondary antibody against mouse MAb conjugated with HRPase. The presence of a band of the expected size was observed (see Figure 13).

m:\specifications\500000\500000\500223clmmjc.doc Example 13: Construction of a synthetic hydroxylated triple helical protein containing two in frame contiguous cassettes of the basic repeat unit for stable expression in yeast.

The construct pFEXB (Figure 10) contains two copies of the basic repeat unit for the triple helical protein and was constructed by introducing the basic subunit into the construct pFEX. This was done through the addition of an XmaI site at the and a BamHI site at the 3'-end and introducing it into the XmaI-BamHI sites of pFEX.

The resulting sequence is

NSGAM-GAPGAP-GGKGDAGAPGERGPP-GLAGAPGLR-

GGAGPPGPEGGKGAAGPPGPP-GGP-GDA-GGKGDAGAPGERGPP-GLAGAPG

R-GGAGPPGPEGGKGAAGPPGPP-GGS-GPVDPR with the underlined sequences derived from the flanking restriction sites and the region in italics arising from the vector sequence at the 3'-end. The basic repeat unit is shown in bold. Yeast were 15 transformed with the construct and the expressed protein analysed by Western blot in the manner described in Example 12. Results are shown in Figure 13.

Example 14: Construction of a synthetic hydroxylated triple helical protein Scontaining two cassettes of the basic repeat unit separated by a 4 amino acid 20 interruption of the helical regions for stable expression in yeast.

The construct pFEXS (Figure 11) contains two copies of the basic repeat unit for the triple helical protein interrupted by a 4 amino acid insertion between the repeat helical domains. pFEXS was constructed by introducing a second copy of the basic 25 repeat subunit into the construct pFEX, using the addition of an BamHI site at the end and a SacII site at the 3' end. This fragment was introduced between the BamHI and SaclI sites of pFEX. The resulting sequence is NSGAM-GAPGAP-GGKGDA (GAPGERG r PP,-GT LAAPTLR---T CGGAGPPGPEGGKGAAGPPGPP-GRS-IDGS- GGKGDAGAPGERGPP-GLAGAPGLR-GGAGPPGPEGGKGAAGPPGPP-GPP with the underlined sequences delineating the 4 amino acid interruption to the Gly-X-Y sequence between the 2 helical region repeats. Yeast were transformed with the construct and the expressed protein analysed by Western blot in the manner described in Example A. Results are shown in Figure 13.

m:\specifications\500000\500000\500223clmmjc.doc Example 15: Construction of a synthetic hydroxylated triple helical protein containing three contiguous cassettes of the basic triple helical repeat unit for stable expression in yeast.

The construct pFEXBS (Figure 14) contains three copies of the basic repeat unit for the triple helical protein and was constructed by introducing the basic subunit into the construct pFEXB. This was done through the addition of a BamHI site at the and a SacII site at the 3' end and introducing it into the BamHI-SacII sites of pFEXB.

The resulting sequence is NSGAM-GAPGAP-GGKGDAGAPGERGPP-GLA

GAPGLR-GGAGPPGPEGGKGAAGPPGPP-GGP-GDA-GGKGDAGAPGERGPP-

GLAGAPGLR-GGAGPPGPEGGKGAAGPPGPP-GGS-GDA-GGKGDAGAPGER

GPP-GLAGAPGLR-GGA GPPGPEGGKGAAGPPGPP GPP with the underlined sequences derived from the linker regions regions generated from the flanking restriction sites used to clone each repeat. The basic repeat unit is shown in bold.

The EcoRI -REPEAT-XmaI fragment cloned in frame with the FLAG-TAG in YEpFlagl to produce pFEX was generated by PCR using primer pair aattcggtgccatggggtgcacctggagctccagga-3' [up] [primer 6772] and ccgggagcaccaggtggcccaggaggga-3' [down] [primer 6771]; the XmaI-REPEAT-BamHI PCR fragment introduced into pFEX to give ppFEXB was generated using the primer 20 pair 5'-tcccc ccggg gatgccggtgcaacctggagctccaa-3' [up] [primer 6775] and gatccaccaggtggcccaggaggacc-3' [down] [primer 6774]; the BamHI-REPEAT-SacII PCR fragment cloned into pFEX to give pFEXS was generated using primer pair gatccggtgcacctggagctccagga-3' [up][primer 6773] and cgcggttaaggtggcccaggaggaccagca-3' [down] [primer 6776] and the PCR fragment 25 comprising the BamHI-REPEAT-SacII introduced into pFEXB to give pFEXBS was generated using the primer pair pair 5'-ccgg gatccggtgcacctggagctccagga-3' [up] [primer 6773] and 5'-ctccc cgcggttaaggtggcccaggaggaccagca- 3 [down] [primer 6776].

Yeast were transformed with the construct and the expressed protein analysed by Western blot in the manner described in Example 12. Results are shown in Figure m:\specifications\500000\500000\500223clmmjc.doc 26 References: Ala-Kokko, L. et al. (1989) Biochem J. 260, 509-516 Amakasu, H. et al. (1993) Genetics 134, 675-683 D'Alessio, M. et al. (1988) Gene 67, 105-115 de Wet, W. et al. (1987) J. Biol. Chem. 262, 16032-16036.

*Cheah, K.S.E. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 2555-2559 Cohen D et al., (1993) Nature 366, 698-701.

15 Gietz, R.D. Sugino, A. (1988) Gene 74, 527-534 Glattauer, V. et al. (1997) Biochem. J. 323, 45-49.

Goff, G. et al (1984) Gene 25, 179-188 Helaakoski, T. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 4392-4396 Heuterspreute, M. et al. (1985) Gene 34, 363-366 25 Hitzeman, R.A. et al (1981) Nature 293, 717-722 Hu, G.Z. Ronne, H. (1994) Nucleic Acid Res. 22, 2740-2743.

Johnston, S.A. Hopper, J.E. (1982) Proc. Natl. Acad. Sci. USA 79, 6971- 6975.

Kuhn RM Ludwig RA, (1994) Gene 141, 125-127.

Larin Z et al., (1996) Nucleic Acid Research 24 No. 21, 4192-4196.

Macreadie, I.G. et al (1989) Plasmid 21, 147-150 Macreadie, I.G. et al. (1994) Biotechnol. Applied Biochem. 19, 265-269 Mahadevan, S. Struhl, K. (1990) Mol. Cell Biol. 10, 4447-4455, 786 Miller, E.J. Rhodes, R.K. (1982) Meth. Enzymol. 82, 33-64.

Pihlajaniemi, T. et al., (1987) EMBO J 6, 643-649.

St. John, T.P. Davis R.W. (1981) J. Mol. Biol. 152, 285-316 Thukral, S.K. et al (1991) Mol. Cell Biol. 11, 699-704 Trelstad, R.L. (1982) Native collagen fractionation. In, Immunochemistry of 15 the Extracellular Matrix Vol. 1 Furthmayr, ed.) CRC Press, 31-41.

Tuite, M.F. et al (1982) EMBO J. 1, 603-608.

Westerhausen, A et al. (1991) Matrix 11, 375-379 i Werkmeister, J.A. Ramshaw, J.A.M. (1991) Biochem. 274, 895-898.

Wiseman, A. (1991) Genetically engineered proteins and enzymes from yeast: production control. Ellis Horwood, New York.

It will be appreciated by persons skilled in the art that numerous variations and/or mnodifications my he made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (46)

1. A method of producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: a first nucleotide sequence encoding prolyl 4-hydroxylase a-subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase P- subunit (P4Hp) operably linked to a promoter sequence functional in said yeast host cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following 15 formula: (A)I-(B)m-(GlyXY)n-(C)o-(D)p, wherein GlyXY represents a triple helical forming repeating sequence Swherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino 20 acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y in the triple helical forming repeating sequence (GlyXY)n is proline, wherein A and D each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, wherein B and C each represent a non-collagenous polypeptide or peptide domain which does not comprise a triple helical forming repeating sequence VV wherein n is an integer of from 2 to 1500, wherein each of 1, m, o and p are selected from 0 and 1, with the proviso that at least one of m and o is 1, and when m is 1 and o is 0, 1 must be 1, and o is 1 and m is 0, then p must be 1, culturing the resulting yeast host cell of step under conditions suitable to express said P4Ha, said P4HP and said polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein, m:\specifications\500000\500000\500223clmmjc.doc wherein during culturing in step each of said first nucleotide sequence molecule, said second nucleotide sequence and said product-encoding nucleotide sequence(s) are replicated, stably retained and segregated by the yeast host cell.

2. A method of claim 1, wherein expression of said P4Ha and said P4HP is controlled in a coordinated manner by a bidirectional promoter sequence.

3. A method according to claim 2, wherein the bidirectional promoter sequence is the yeast GAL 1-10 promoter sequence.

4. A method according to any one of the preceding claims, wherein the first and second nucleotide sequences are of avian or mammalian origin. 15 5. A method according to claim 4, wherein the first and second nucleotide sequences are of human origin.

6. A method according to any one of claims 1 to 5 wherein the second and product- encoding nucleotide sequences encode secretion signals such that expressed P4H and 20 product polypeptide(s) or peptide(s) are secreted.

7. A method according to any one of claims 1 to 5, wherein the second and product-encoding nucleotide sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s).

8. A method according to any one of claims 1 to 6, wherein the first, second and product-encoding sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s) and one or two high copy number vector(s).

9. A method according to claim 7 or 8, wherein the one or more vector(s) including a CEN sequence(s) are selected from YAC vectors. A method according to claim 8, wherein the one or two high copy number vector(s) are selected from YEp plasmids. m:\specifications\500000\500000\500223clmmjc.doc

11. A method according to claim 10, wherein the first, second and product-encoding nucleotide sequences are present on a single YAC vector.

12. A method according to any one of the preceding claims, wherein the yeast host cell is selected from the genus Kluveromyces, Saccharomyces, Schizosaccharomyces, Yarrowia and Pichia.

13. A yeast host cell which produces a hydroxylated triple helical protein upon culturing, wherein said yeast host cell comprises: a first nucleotide sequence encoding prolyl 4-hydroxylase a- subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, a second nucleotide sequence encoding prolyl 4-hydroxylase P- subunit (P4Hp) operably linked to a promoter sequence functional in said yeast host 15 cell, and one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: *0 wherein GlyXY represents a triple helical forming repeating sequence, wherein Gly represents glycine, wherein X and Y, which may be the same or different, each represent an amino acid, and wherein the identity of each amino acid represented by X and Y may var from CGvYV trinlpt to GlvYX trinlt hut wherein at least one Y in the triple helical forming repeating sequence (GlyXY)n is proline, wherein A and D each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY)n, wherein B and C each represent a non-collagenous polypeptide or peptide domain which does not comprise a triple helical forming repeating sequence (GlyXY)n, wherein n is an integer of from 2 to 1500, m:\specifications\500000\500000\500223clmmjc.doc wherein each of 1, m, o and p are selected from 0 and 1, with the proviso that at least one of m and o is 1, and when m is 1 and o is 0, 1 must be 1, and when o is 1 and m is 0, then p must be 1, and wherein upon culturing of said yeast host cell, each of said first nucleotide sequence, said second nucleotide sequence and said product encoding nucleotide sequence(s) are replicated, stably retained and segregated by said yeast host cell.

14. A yeast host cell according to claim 13, wherein expression of said P4Ha and said P4HP is controlled in a coordinated manner by a bidirectional promoter. A yeast host cell according to claim 14, wherein the bidirectional promoter sequence is the yeast GAL 1-10 promoter sequence. 15 16. A yeast host cell according to any one of claims 13 to 15, wherein the first and second nucleotide sequences are of avian or mammalian origin.

17. A yeast host cell according to claim 16, wherein the first and second nucleotide sequences are of human origin.

18. A yeast host cell according to any one of claims 13 to 17, wherein the second and product-encoding nucleotide sequences encode secretion signals such that expressed P4H and product polypeptide(s) or peptide(s) are secreted. 25 19. A yeast host cell according to any one of claims 13 to 18, wherein the first, second and product-encoding nucleotide sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including CEN sequence(s). A yeast host cell according to any one of claims 13 to 18, wherein the first, second and product-encoding sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s) and one or two high copy number vector(s).

21. A yeast host cell according to claim 19 or 20, wherein the one or more vector(s) including a CEN sequence(s) are selected from YAC vectors. m:\specifications\500000\500000\500223clmmjc.doc

22. A yeast host cell according to claim 20, wherein the one or two high copy number plasmid(s) are selected from YEp plasmids.

23. A yeast host cell according to claim 19, wherein the first, second and product- encoding nucleotide sequences are present on a single YAC vector.

24. A yeast host cell according to any one of claims 13 to 23, wherein the yeast host cell is selected from the genus Kluveromyces, Saccharomyces, Schizosaccharomyces, Yarrowia and Pichia. A method according to any one of claims 1 to 12, wherein the triple helical forming repeating sequence (Gly X Y)n includes at least one integrin binding site.

26. A method according to claim 25, wherein the integrin binding site comprises the 15 amino acid sequence: GLAGAPGLR. o*

27. A yeast host cell according to any one of claims 13 to 24, wherein the triple helical forming repeating sequence (GlyXY)n includes at least one integrin binding site. 20 28. A yeast host cell according to claim 27, wherein the integrin binding site comprises the amino acid sequence: GLAGAPGLR.

29. A triple helical protein produced in accordance with the method of any one of 2 claims 1 to 12, 25 or 26. A biomaterial or therapeutic product comprising a triple helical protein produced in accordance with the method of any one of claims 1 to 12, 25 or 26.

31. A method of producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of: introducing into a suitable yeast host cell: a first nucleotide sequence encoding prolyl 4-hydroxylase a-subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase 13- subunit (P4HP) operably linked to a promoter sequence functional in said yeast host cell, and m:\specifications\500000\500000\500223clmmjc.doc (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polypeptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i wherein; wherein GlyXY represents a triple helical forming repeating sequence; E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each *i 15 independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, .Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary 20 from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY),; B and C, which may be the same or different, each represent a polypeptide or nart;iAP trnmn ixrhilh ic hptprnlon1 tn rn1llaopn nrnteins and which does not comprise a triple helical forming repeating sequence (GlyXY)n wherein each of m and o is 1 and each of I and p are selected from 0 and 1; and culturing the resulting yeast host cell of step under conditions suitable to express said P4Ha, P4HP and said synthetic polypeptide(s) or peptide(s), to produce said hydroxylated triple helical protein; and wherein during culturing in step each of said first nucleotide sequence, said second nucleotide sequence and said product-encoding nucleotide m:\specifications\500000\500000\500223clmmjc.doc sequence(s) are replicated, stably retained and segregated by the yeast host cell.

32. A method according to claim 31, wherein domain Z comprises no more than to 300 GlyXY triplets.

33. A method according to claim 31 or 32, wherein in domain Z, (GlyXY)i has an amino acid length which is at least three times greater than the combined amino acid length of E and F.

34. A method according to any one of claims 31 to 33, wherein expression of the said P4Ha subunit and said P4HP subunit is controlled in a coordinated manner by a bidirectional promoter. S**i 15 35. A method according to claim 34, wherein the bidirectional promoter sequence is the yeast GAL 1-10 promoter sequence.

36. A method according to any one of the preceding claims, wherein the first and Ssecond nucleotide sequences are of avian or mammalian origin.

37. A method according to claim 36, wherein the first and second nucleotide sequences are of human origin.

38. A method according to any one of claims 33 to 37 wherein the second and 25 product-encoding nucleotide sequences encode secretion signals such that expressed P4H and product polypeptide(s) or peptide(s) are secreted.

39. A method according to any one of claims 33 to 37, wherein the second and product-encoding nucleotide sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s). A method according to any one of claims 33 to 38, wherein the first, second and product-encoding sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s) and one or two high copy number vector(s). m:\specifications\500000\500000\500223clmmjc.doc

41. A method according to claim 39 or 40, wherein the one or more vector(s) including a CEN sequence(s) are selected from YAC vectors.

42. A method according to claim 40, wherein the one or two high copy number vector(s) are selected from YEp plasmids.

43. A method according to claim 42, wherein the first, second and product-encoding nucleotide sequences are present on a single YAC vector.

44. A method according to any one of the preceding claims, wherein the yeast host cell is selected from the genus Kluveromyces, Saccharomyces, Schizosaccharomyces, Yarrowia and Pichia. i

45. A yeast host cell capable of producing a hydroxylated triple helical protein upon 15 culturing, said yeast host cell including: S* a first nucleotide sequence encoding prolyl 4-hydroxylase a-subunit (P4Ha) operably linked to a promoter sequence functional in said yeast host cell, (ii) a second nucleotide sequence encoding prolyl 4-hydroxylase P- subunit (P4HP) operably linked to a promoter sequence functional in said yeast host 20 cell, and (iii) one or more product-encoding nucleotide sequence(s) encoding a polypeptide(s) or peptide(s) operably linked to a promoter sequence functional in said yeast host cell, wherein said polyp~ptide(s) or peptide(s) is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said :g \25 polypeptide(s) or peptide(s) is a synthetic polypeptide(s) or peptide(s) represented by the following formula: (B)m (C)o wherein; Z is a domain comprising two or more repeat units of the formula: (GlyXY)i wherein; wherein GlyXY represents a triple helical forming repeating sequence; m:\specifications\500000\500000\500223clmmjc.doc 36 E and F represent sequences of one or more amino acids, which sequences may vary from repeat unit to repeat unit, and for each repeat unit q and r are each independently selected from 0 and 1, and i is 1 such that domain Z comprises 2 to 1500 GlyXY triplets, Gly represents glycine, and X and Y, which may be the same or different, represent an amino acid, and wherein the identity of each amino acid represented by X and Y may vary from GlyXY triplet to GlyXY triplet, but wherein at least one Y of the (GlyXY)i sequence must be proline, A and D, which may be the same or different, each represent a polypeptide or peptide domain which optionally comprises a triple helical forming repeating sequence (GlyXY),, B and C, which may be the same or different, each represent a polypeptide or peptide domain which is heterologous to collagen proteins and which does 15 not comprise a triple helical forming repeating sequence (GlyXY)n, wherein each of m and o is 1 and each of 1 and p are selected from 0 and 1, and; wherein upon culturing of said yeast host cell, each of said first nucleotide sequence, said second nucleotide sequence and said product-encoding nucleotide sequence(s) are replicated, stably retained and segregated by the 20 yeast host cell.

46. A yeast host cell according to claim 45, wherein domain Z comprises no more than 10 to 300 GlyXY triplets.

47. A yeast host cell according to claim 45 or 46, wherein in domain Z, (GlyXY)i has an amino acid length which is at least three times greater than the combined amino acid length of E and F.

48. A yeast host cell according to any one of claims 45 to 47, wherein expression of the said P4Ha subunit and said P4H3 subunit is controlled in a coordinated manner by a bidirectional promoter.

49. A yeast host cell according to claim 48, wherein the bidirectional promoter sequence is the yeast GALl-10 promoter sequence. m:\specifications\500000\500000\500223cmmjc.doc 23/02 '05 14:34 FAX 61 3 9663 3099 FB RICE CO. __00O5 37 A yeast host cell according to any one of claims 47 to 49, wherein the first and second nucleotide sequences are of avian or mammalian origin.

51. A yeast host cell according to claim 50, wherein the first and second nucleotide sequences are of human origin.

52. A yeast host cell according to any one of claims 47 to 51, wherein the second and product-encoding nucleotide sequences encode secretion signals such that expressed P4H and product polypeptide(s) or peptide(s) are secreted.

53. A yeast host cell according to any one of claims 47 to 52, wherein the first, second and product-encoding nucleotide sequences are introduced to the yeast host cell i such that they are present on one or more vector(s) including CEN sequence(s). 15 54. A yeast host cell according to any one of claims 47 to 52, wherein the first, *I second and product-encoding sequences are introduced to the yeast host cell such that they are present on one or more vector(s) including a CEN sequence(s) and one or two 20 high copy number vector(s). 20 55. A yeast host cell according to claim 53 or 54, wherein the one or more vector(s) including a CEN sequence(s) are selected from YAC vectors. S56. A yeast host cell according to claim 54, wherein the one or two high copy S* number plasmid(s) are selected from YEp plasmids.

57. A yeast host cell according to claim 53, wherein the first, second and product- encoding nucleotide sequences are present on a single YAC vector,

58. A yeast host cell according to any one of claims 47 to 57, wherein the yeast host ,n 4..n Vminrx.n...-f--, rom Sr r.hiznxaccharomvces, Yarrowia and Pichia.

59. A hydroxylated triple helical protein produced in accordance with the method of any one of claims 31 to 44. m;\specificatlons\5OOOOO\50000\500 22 3Imb1 6mjc.doc COMS ID No: SBMI-01134375 Received by IP Australia: Time 14:33 Date 2005-02-23 23/02 '05 14:34 FAX 61 3 9663 3099 FB RICE CO. 38 A hydroxylated triple helical protein of claim 59, wherein domain Z comprises no more than 10 to 300 GlyXY triplets. ao006

61. A hydroxylated triple helical protein according to claim 58 or 59, wherein in domain Z, (GlyXY)i has an amino acid length which is at least three times greater than the combined amino acid length of E and F.

62. A biomaterial or therapeutic product comprising an hydroxylated triple helical according to any one of claims 59 to 61. Dated this eighteenth day of February 2005 9 .9 9. 9909 0 *900 9 9 9. 9 9 9 @9 *9 Commonwealth Scientific and Industrial Research Organisation Patent Attorneys for the Applicant: F B RICE CO m:\specifications\500000\5000o0\ 22-3Imbl 6 mjc.doc COMS ID No: SBMI-01134375 Received by IP Australia: Time 14:33 Date 2005-02-23