Method for producing, in yeast, a hydroxylated triple helical protein, and yeast host cells useful in said method

Abstract

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 (PH4), in a manner wherein the introduced DNA sequences are replicated, stably retained and segregated by the yeast host cells.

1 . A method of producing, in yeast, a hydroxylated triple helical protein, said method comprising the steps of:
 (A) introducing into a suitable yeast host cell:
 (i) a first DNA molecule comprising a DNA sequence encoding prolyl 4-hydroxylase I-subunit (P4HI) operably linked to a promoter functional in said yeast host cell, 
 (ii) a second DNA molecule comprising a DNA sequence encoding prolyl 4-hydroxylase 4-subunit (P4Hl) operably linked to a promoter functional in said yeast host cell, and 
 (iii) a third DNA molecule comprising a DNA sequence encoding a polypeptide or peptide operably linked to a promoter functional in said yeast host cell, wherein said polypeptide or peptide is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide or peptide is a synthetic polypeptide or peptide represented by the following formula:
   (A) 1 −(B) m −[Z]−(C)O−(D) p , 
 
   
       wherein;
 Z is a domain comprising two or more repeat units of the formula:
   [(E) q −(GlyXY) i −(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 
 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) n , and l 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; and 
 (B) culturing the resulting yeast host cell of step (A) under conditions suitable to express said P4HI and P4Hl and said synthetic polypeptide or peptide, to produce said hydroxylated triple helical protein; 
 wherein during culturing in step (B), each of said first DNA molecule, said second DNA molecule and said third DNA molecule are replicated, stably retained and segregated by the yeast host cell. 
 
     
     
         2 . The method of  claim 1 , wherein domain Z comprises no more than 10 to 300 GlyXY triplets. 
     
     
         3 . The method of  claim 1 , 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. 
     
     
         4 . The method of  claim 1 , wherein expression of the said P4HI subunit and said and P4Hl subunit is controlled in a coordinated manner by a bidirectional promoter. 
     
     
         5 . The method of  claim 4 , wherein said bidirectional promoter is yeast GAL1-10 promoter sequence. 
     
     
         6 . The method of  claim 1 , wherein said P4HI subunit is an avian P4HI subunit or a mammalian P4HI subunit, and said P4Hl subunit is an avian P4Hl subunit or a mammalian P4Hl subunit. 
     
     
         7 . The method of  claim 6 , wherein said mammalian P4HI subunit is human P4HI subunit, and said mammalian P4Hl subunit is human P4H& subunit. 
     
     
         8 . The method of  claim 1 , wherein each of said second DNA molecule and said third DNA molecule further comprise a DNA sequence encoding a secretion signal such that said P4Hl and said polypeptide(s) or peptide(s) are expressed and secreted by said yeast host cell. 
     
     
         9 . The method of  claim 1 , wherein in step (A) each of said first DNA molecule, said second DNA molecule and said third DNA molecule is present on a vector, which may be the same or different, comprising a CEN sequence. 
     
     
         10 . The method of  claim 1 , wherein in step (A), at least one of said first DNA molecule, said second DNA molecule and said third DNA molecule is present on a vector, which may be the same or different, and comprises a CEN sequence, and at least one of said first DNA molecule, said second DNA molecule and said third DNA molecule is present on a high copy number vector, which may be the same or different. 
     
     
         11 . The method of  claim 9 , wherein said vector comprising a CEN sequence is a YAC vector. 
     
     
         12 . The method of  claim 10 , wherein said vector comprising a CEN sequence is a YAC vector. 
     
     
         13 . The method of  claim 10 , wherein said high copy number vector is a YEp plasmid. 
     
     
         14 . The method of  claim 11 , wherein said first DNA molecule, said second DNA molecule and said third DNA molecule are present on the same YAC vector. 
     
     
         15 . The method of  claim 1 , wherein said yeast host cell is a member of a genus selected from the group consisting of  Kluveromyces, Saccharomyces, Schizosaccharomyces, Yarrowia  and  Pichia.    
     
     
         16 . A yeast host cell capable of producing a hydroxylated triple helical protein upon culturing, said yeast host cell including:
 (i) a first DNA sequence encoding prolyl 4-hydroxylase I-subunit (P4HI) operably linked to a promoter functional in said yeast host cell,   (ii) a second DNA sequence encoding prolyl 4-hydroxylase l-subunit   (P4Hl) operably linked to a promoter functional in said yeast host cell, and   (iii) a third DNA sequence encoding a polypeptide or peptide operably linked to a promoter functional in said yeast host cell, wherein said polypeptide or peptide is one which, when hydroxylated, forms said hydroxylated triple helical protein, and wherein said polypeptide or peptide is a synthetic polypeptide or peptide represented by the following formula:
   (A) i −(B) m −[Z]−(C) o −(D) p , 
   
       wherein;
 Z is a domain comprising two or more repeat units of the formula:
   [(E) q −(GlyXY) i −(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 
 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) n , and l 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; and 
 wherein each of said first DNA sequence, said second DNA sequence and said third DNA sequence are replicated, stably retained and segregated by the yeast host cell. 
 
     
     
         17 . A synthetic recombinant hydroxylated triple helical protein comprising a polypeptide or peptide having one or more non-collagenous, non-triple helical domains which is a synthetic polypeptide or peptide selected from the group consisting of collagens, collagen fragments and collagen-like proteins represented by the following formula:
   (A) l −(B) m −[Z]−(C) o −(D) p ,   
       wherein:
 Z is a domain comprising two or more repeat units of the formula:
   [(E) q −(GlyXY) i −(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 
 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) n , wherein n is 2 to 1500 and l 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. 
 
     
     
         18 . The synthetic recombinant hydroxylated triple helical protein of  claim 17 , wherein domain Z comprises 10 to 300 GlyXY triplets. 
     
     
         19 . The synthetic recombinant hydroxylated triple helical protein of  claim 17 , 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. 
     
     
         20 . A biomaterial or therapeutic product comprising said synthetic recombinant hydroxylated triple helical protein of  claim 18 , wherein the biomaterial or therapeutic product is formed from a member selected from the group consisting of collagens, natural and synthetic collagen fragments, and natural and synthetic collagen-like proteins. 
     
     
         21 . A biomaterial or therapeutic product comprising said synthetic recombinant hydroxylated triple helical protein of  claim 19 , wherein the biomaterial or therapeutic product is formed from a member selected from the group consisting of collagens, natural and synthetic collagen fragments, and natural and synthetic collagen-like proteins. 
     
     
         22 . The synthetic recombinant hydroxylated triple helical protein of  claim 17 , wherein said protein comprises a biologically active domain(s). 
     
     
         23 . A synthetic recombinant hydroxylated triple helical protein consisting essentially of a polypeptide or peptide having one or more non-collagenous, non-triple helical domains which is a synthetic polypeptide or peptide selected from the group consisting of collagens, collagen fragments and collagen-like proteins represented by the following formula:
   (A) 1 −(B) m −[Z]−(C) o −(D) p ,   
       wherein:
 Z is a domain comprising two or more repeat units of the formula:
   [(E) q −(GlyXY) i −(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 
 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) n , wherein n is 2 to 1500 and l 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.