Highly stable recombinant yeasts for the production of recombinant proteins

Abstract

Novel recombinant yeasts are disclosed which are highly stable in a complex medium. Said yeasts are of the Kluyveromyces genus in which an essential gene is nonfunctional, containing a vector bearing a functional copy of said gene.

We claim:  
     
       1. Yeast of the Kluyveromyces genus exhibiting the Pgk −  phenotype. 
     
     
       2. K. lactis yeast FB05D. 
     
     
       3. A host/vector pair which is stable in a complex medium, wherein the host is a yeast of the Kluyveromyces genus lacking a functional phosphoglycerate kinase gene, and wherein the vector comprises a functional copy of the phosphoglycerate kinase gene. 
     
     
       4. The host/vector pair according to  claim 3 , wherein the functional copy of the phosphoglycerate kinase gene present in the vector is placed under the control of a weak promoter or a defective promoter, or is completely free of a promoter. 
     
     
       5. The host/vector pair according to  claim 3 , wherein the host is selected from the group consisting of Kluyveromyces lactis and Kluyveromyces fragilis. 
     
     
       6. The host/vector pair according to  claim 3 , wherein the vector additionally comprises a DNA sequence encoding a desired protein, and signals permitting expression of said sequence. 
     
     
       7. The host/vector pair according to  claim 6 , wherein the protein is selected from the group consisting of enzymes, blood derivatives, insulin, variants of insulin, lymphokines, growth factors, apolipoproteins, antigenic polypeptides for the production of vaccines, viral receptors, and fusion polypeptides comprising an active part fused to a stabilising part. 
     
     
       8. The host/vector pair according to  claim 7 , wherein said enzymes are selected from the group consisting of superoxide dismutase, catalase, amylases, lipases, amidases, and chymosin. 
     
     
       9. The host/vector pair according to  claim 7 , wherein said blood derivatives are selected from the group consisting of serum albumin, variants of serum albumin, alpha-globin, beta-globin, factor VII, factor IX, non Willebrand factor (vWF), biologically active fragments of vWF, fibronectin and 1-alpha-antitrypsin. 
     
     
       10. The host/vector pair according to  claim 7 , wherein said lymphokines are selected from the group consisting of the interleukins, interferons, and colony stimulation factors. 
     
     
       11. The host/vector pair according to  claim 7 , wherein said colony stimulation factors are selected from the group consisting of G-CSF, GM-CSF, M-CSF, TNF, and TRF. 
     
     
       12. The host/vector pair according to  claim 7 , wherein said growth factors are selected from the group consisting of growth hormone, erythropoietin, FGF, EGF, PDGF, and TGF. 
     
     
       13. The host/vector pair according to  claim 7 , wherein said antigenic polypeptides are selected from the group consisting of antigens from a hepatitis virus, cytomegalovirus virus, an Epstein-Barr virus, and a herpes virus. 
     
     
       14. A method for producing proteins, said method comprising culturing the host/vector pair of  claim 6  and recovering the proteins produced, wherein the protein is selected from the group consisting of enzymes, blood derivatives, insulin, variants of insulin, lymphokines, growth factors, apolipoproteins, antigenic polypeptides for the production of vaccines, viral receptors, and fusion polypeptides comprising an active part fused to a stabilising part. 
     
     
       15. The method according to  claim 14 , wherein said enzymes are selected from the group consisting of superoxide dismutase, catalase, amylases, lipases, amidases, and chymosin. 
     
     
       16. The method according to  claim 14 , wherein said blood derivatives are selected from the group consisting of serum albumin, variants of serum albumin, alpha-globin, beta-globin, factor VIII, factor IX, non Willebrand factor (vWF), biologically active fragments of vWF, fibronectin and 1-alpha-antitrypsin. 
     
     
       17. The method according to  claim 14 , wherein said lymphokines are selected from the group consisting of the interleukins, interferons, and colony stimulation factors. 
     
     
       18. The method according to  claim 14 , wherein said colony stimulation factors are selected from the group consisting of G-CSF, GM-CSF, M-CSF, TNF, and TRF. 
     
     
       19. The method according to  claim 14 , wherein said growth factors are selected from the group consisting of growth hormone, erythropoietin, FGF, EGF, PDGF, and TGF. 
     
     
       20. The method according to  claim 14 , wherein said antigenic polypeptides are selected from the group consisting of antigens from a hepatitis virus, a cytomegalovirus virus, an Epstein-Barr virus, and a herpes virus. 
     
     
       21. The host/vector pair according to  claim 6  wherein the DNA sequence additionally comprises signals enabling secretion of the protein. 
     
     
       22. The host/vector pair according to  claim 6  wherein the signals enabling the expression of the second  DNA sequence are selected from the group consisting of transcription promoters and terminators. 
     
     
       23. The host/vector pair according to  claim 22 , wherein said promoters are selected from the group consisting of promoters of yeast genes, and modified promoters of yeast genes. 
     
     
       24. A method for producing a recombinant protein, comprising culturing the host/vector pair of  claim 6  and recovering the protein produced. 
     
     
       25. The method according to claim  14  for producing  16  wherein said blood derivatives are human serum albumin, its precursors and variants. 
     
     
       26. An expression vector comprising a first DNA sequence encoding functional Kluyveromyces phosphoglycerate kinase, a second DNA sequence encoding a pharmaceutical protein, and a third DNA sequence encoding signals permitting expression of said second DNA sequence. 
     
     
       27. A vector selected from the group consisting of pYG1023, pYG1033 and pYG1033ΔSfiI.