Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences

Abstract

A yeast alpha-factor expression system is provided comprised of a truncated leader sequence, containing the alpha-factor signal peptide and one glycosylation site, linked by a processing site to a non-yeast protein-encoding sequence.

I claim:  
     
       1. A yeast cell comprising a DNA construct that provides for the expression and secretion of a non-yeast protein, said DNA construct comprising 
       a yeast recognized transcription initiation sequence, linked 5′ to  
       a coding sequence under the control of both said yeast recognized transcription initiation sequence and  
       a yeast-recognized termination sequence, said yeast-recognized termination sequence being 3′ to said coding sequence,  
       wherein said coding sequence encodes a precursor polypeptide comprised of a leader sequence and said non-yeast protein linked by a processing site that provides for the cleavage of said non-yeast protein from said precursor polypeptide,  
       wherein said leader sequence is about the first 25 to about the first 50 N-terminal residues of a yeast alpha-factor leader polypeptide,  
       comprises a single yeast alpha-factor precursor glycosylation site and comprises a single peptide of a yeast alpha-factor precursor comprising the first about 19 to about 23 N-terminal residues of said alpha-factor precursor.  
     
     
       2. The cell of claim  1  wherein said non-yeast protein is a mammalian protein. 
     
     
       3. The cell of claim  2  wherein said mammalian protein is a precursor of human insulin. 
     
     
       4. The cell of claim  3  wherein said precursor of human insulin is human proinsulin. 
     
     
       5. The cell of claim  3   1 wherein said precursor of human insulinnon- yeast protein  comprises insulin a chain and insulin b chain linked by a yeast-recognized processing site cleaved in vivo. 
     
     
       6. The cell of claim  5  wherein said processing site is cleaved by the KEX2 gene product of Saccharomyces. 
     
     
       7. The cell of claim  2  wherein said mammalian protein is insulin-like growth factor I. 
     
     
       8. The cell of claim  1  wherein said yeast cell is from the genus Saccharomyces. 
     
     
       9. The cell of claim  8  wherein said yeast cell is  S. cerevisiae.    
     
     
       10. The cell of claim  8  wherein yeast α-factor precursor is  S. cerevisiae  MFα1. 
     
     
       11. The cell of claim  1  wherein said leader sequence is about 28 to about 40 N-terminal residues of said precursor polypeptide. 
     
     
       12. The cell of claim  1  wherein said leader sequence is about 35 contiguous N-terminal residues of a yeast alpha-factor precursor polypeptide. 
     
     
       13. A double-stranded DNA molecule comprising a region encoding a precursor polypeptide secretable by a yeast host, said region, with reference to one of the strands, having  comprising the structure: 
       
         
           5′-AF-CHO-X n -S-Gene*-3′ 
         
       
       wherein 
       AF encodes a yeast alpha-factor signal peptide;  
       CHO encodes a yeast alpha-factor precursor  glycosylation site comprising the amino acid sequence Asn- Y - Y′ wherein Y is any amino acid and Y′ is Thr or Ser;   
       X n  encodes a spacer polypeptide of n amino acids in length that does not contain a glycosylation site or a processing site that provides for cleavage of said precursor polypeptide in vivo by yeast;  
       n is an integer from 0 to 30;  
       Gene* encodes a non-yeast protein; and  
       S encodes a processing site that provides for cleavage of said precursor polypeptide.  
     
     
       14. The DNA molecule of claim  13  wherein AF encodes a polypeptide of about 19 to 23 amino acids in length. 
     
     
       15. The DNA molecule of claim  13  wherein n is an integer from about 0 to about 20. 
     
     
       16. The DNA molecule of claim  13  wherein n is an integer from about 0 to about 10. 
     
     
       17. The DNA molecule of claim  13  wherein n is an integer from about 3 to about 10. 
     
     
       18. The DNA molecule of claim  13  wherein said yeast host is a Saccharomyces. 
     
     
       19. The DNA molecule of claim  13  wherein said yeast α-factor signal peptide is a Saccharomyces signal peptide. 
     
     
       20. The DNA molecule of claim  13  wherein S encodes a processing site recognized in vivo by said yeast host. 
     
     
       21. The DNA molecule of claim  20  wherein S encodes a dipeptide recognized by the KEX2 endopeptidase. 
     
     
       22. The DNA molecule of claim  21  wherein said dipeptide is 5′-Lys-Arg-3′ or 5′-Arg-Arg-3′. 
     
     
       23. The DNA molecule of claim  13  comprising a replicon. 
     
     
       24. The DNA molecule of claim  23  wherein said region encoding said precursor polypeptide is under the control of yeast-recognized transcription initiation and termination sequences, and said replicon is a yeast replicon. 
     
     
       25. The DNA molecule of claim  24  wherein said replicon is a plasmid. 
     
     
       26. The DNA molecule of claim  24  wherein said replicon is a chromosome. 
     
     
       27. A DNA molecule comprising a nucleotide sequence that encodes about the first 25 to about the first 50 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 
     
     
       28. The DNA molecule of claim  27  wherein the nucleotide sequence encodes about the first 25 to about the first 40 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 
     
     
       29. The DNA molecule of claim  27  wherein the nucleotide sequence encodes about the first 35 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and not other yeast alpha-factor precursor leader sequence glycosylation site. 
     
     
       30. The DNA molecule of claim  27  wherein the nucleotide sequence encodes about the first 28 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 
     
     
       31. The DNA molecule of claim  27  wherein the nucleotide sequence encodes amino acids 1-25 and 81-83 of a yeast alpha-factor precursor leader sequence. 
     
     
       32. A method for producing a recombinant protein, said method comprising: 
       
         providing a yeast cell as defined in claim  1 , and  
       
         culturing said yeast cell under conditions that provide for expression and secretion of said non - yeast protein.   
     
     
       33. The method of claim  32  wherein said non- yeast protein is a mammalian protein.   
     
     
       34. The method of claim  32  wherein said non- yeast protein is a precursor of human insulin.   
     
     
       35. The method of claim  34  wherein said precursor of human insulin is human proinsulin. 
     
     
       36. The method of claim  34  wherein said precursor of human insulin comprises insulin a chain and insulin b chain linked by a yeast- recognized processing site cleaved in vivo.   
     
     
       37. The method of claim  36  wherein said processing site is cleaved by the KEX 2  gene product of Saccharomyces. 
     
     
       38. The method of claim  33  wherein said mammalian protein is insulin- like growth factor I.   
     
     
       39. The method of claim  32  wherein said yeast cell is from the genus Saccharomyces. 
     
     
       40. The method of claim  39  wherein said yeast cell is S. cerevisiae. 
     
     
       41. The method of claim  39  wherein said yeast alpha- factor leader polypeptide is from S. cerevisiae MFα 1 .   
     
     
       42. The method of claim  32  wherein said leader sequence is about the first  28  to about the first  40  N- terminal amino acid residues of a yeast alpha - factor leader polypeptide.   
     
     
       43. The method of claim  32  wherein said leader sequence is about the first  35  contiguous N- terminal residues of a yeast alpha - factor leader polypeptide.   
     
     
       44. A method of producing a recombinant protein, said method comprising: 
         transforming a yeast cell with a DNA construct that provides for the expression and secretion of a non - yeast protein, wherein said DNA construct comprises the double - stranded DNA molecule as defined in claim  13 , and    
         culturing said transformed yeast cell under conditions that provide for expression and secretion of said non - yeast protein.   
     
     
       45. The method of claim  44  wherein AF encodes a polypeptide of about  19  to  23  amino acids in length. 
     
     
       46. The method of claim  44  wherein n is an integer from about  0  to about  20 . 
     
     
       47. The method of claim  44  wherein n is an integer from about  0  to about  10 . 
     
     
       48. The method of claim  44  wherein n is an integer from about  3  to about  10 . 
     
     
       49. The method of claim  44  wherein said yeast host is a Saccharomyces. 
     
     
       50. The method of claim  44  wherein said yeast alpha- factor signal peptide is a Saccharomyces signal peptide.   
     
     
       51. The method of claim  44  wherein S encodes a processing site recognized in vivo by said yeast host. 
     
     
       52. The method of claim  51  wherein S encodes a dipeptide recognized by the KEX 2  endopeptidase. 
     
     
       53. The method of claim  52  wherein said dipeptide is  5 ′- Lys - Arg -   3 ′ or  5 ′ - Arg - Arg -   3 ′.   
     
     
       54. The method of claim  44  wherein said DNA molecule comprises a replicon. 
     
     
       55. The method of claim  54  wherein said region encoding said precursor polypeptide is under the control of yeast- recognized transcription initiation and termination sequences, and said replicon is a yeast replicon.   
     
     
       56. The method of claim  55  wherein said replicon is a plasmid. 
     
     
       57. The method of claim  55  wherein said replicon is a chromosome. 
     
     
       58. A method of producing a recombinant protein, said method comprising: 
         transforming a yeast cell with a DNA construct that provides for the expression and secretion of a non - yeast protein, wherein said DNA construct comprises the double - stranded DNA molecule as defined in claim  27 , and    
         culturing said transformed yeast cell under conditions that provide for expression and secretion of said non - yeast protein.   
     
     
       59. The method of claim  58  wherein the nucleotide sequence encodes about the first  25  to about the first  40  contiguous N- terminal amino acids of a yeast alpha - factor precursor leader sequence that includes a single yeast alpha - factor precursor leader sequence glycosylation site and no other yeast alpha - factor precursor leader sequence glycosylation site.   
     
     
       60. The method of claim  58  wherein the nucleotide sequence encodes about the first  35  contiguous N- terminal amino acids of a yeast alpha - factor precursor leader sequence that includes a single yeast alpha - factor precursor leader sequence glycosylation site and no other yeast alpha - factor precursor leader sequence glycosylation site.   
     
     
       61. The method of claim  58  wherein the nucleotide sequence encodes about the first  28  contiguous N- terminal amino acids of a yeast alpha - factor precursor leader sequence that includes a single yeast alpha - factor precursor leader sequence glycosylation site and no other yeast alpha - factor precursor leader sequence glycosylation site.   
     
     
       62. The method of claim  59  wherein the nucleotide sequence encodes amino acids  1 - 25  linked to amino acids  81 - 83  of a yeast alpha- factor precursor leader sequence.   
     
     
       63. The method of claim  44  wherein CHO encodes a yeast alpha- factor precursor glycosylation site.   
     
     
       64. The DNA molecule of claim  13  wherein CHO encodes a yeast alpha- factor precursor glycosylation site.   
     
     
       65. The method of claim  49  wherein said yeast host is S. cerevisiae. 
     
     
       66. The method of claim  49  wherein the leader construct AF- CHO - X   n - S is about the first  25  to about the first  40  contiguous N - terminal residues of a yeast alpha - factor signal peptide.   
     
     
       67. The method of claim  49  wherein the leader construct AF- CHO - X   n - S is about the first  28  to about the first  40  contiguous N - terminal residues of a yeast alpha - factor signal peptide.   
     
     
       68. The method of claim  49  wherein the leader construct AF- CHO - X   n - S is about the first  35  to about the first  40  contiguous N - terminal residues of yeast alpha - factor signal peptide.   
     
     
       69. The method of claim  68  wherein said non- yeast protein is a human proinsulin.   
     
     
       70. The method of claim  69  wherein said DNA construct is plasmid pYGAI 7  ( ATCC Accession Number  67597   ). 
     
     
       71. The method of claim  69  wherein said human proinsulin comprises insulin a chain and insulin b chain linked by a yeast- recognized processing site cleaved in vivo by the KEX 2  gene product of Saccharomyces.   
     
     
       72. The method of claim  71  wherein said DNA construct is plasmid pYαf L     7 C 3   ( ATCC Accession Number  67596   ). 
     
     
       73. The method of claim  49  wherein the leader construct AF- CHO - X   n - S is amino acids  1 - 25  linked to amino acids  81 - 83  of a yeast alpha - factor signal peptide.   
     
     
       74. The method of claim  73  wherein said non- yeast protein is insulin - like growth factor I.   
     
     
       75. The method of claim  74  wherein said DNA construct is plasmid pYLUIGFI-   55   ( ATCC Accession Number  67595   ).