Process for production of polypeptides

Abstract

Vectors for producing polypeptides heterologous to prokaryotes are described comprising, along with the polypeptide-encoding nucleic acid, anti-termination nucleic acid that inhibits intragenic transcription termination with a non-lambda promoter therefor and/or nucleic acid encoding a GreA or GreB protein and a promoter therefor. Also described are processes for producing a heterologous polypeptide in prokaryotic host cells utilizing such elements to improve the quality and/or quantity of heterologous polypeptide produced.

1 . A vector for producing a polypeptide heterologous to prokaryotic cells comprising (1) anti-termination nucleic acid that inhibits intragenic transcription termination with a non-lambda promoter therefor, and (2) RNA encoding the polypeptide with a non-lambda promoter therefor, wherein an RNA recognition site for binding anti-termination protein produced from the nucleic acid is located 5′ of the RNA encoding the polypeptide.  
     
     
         2 . The vector of  claim 1  further comprising nucleic acid encoding a GreA or GreB protein with a promoter therefor.  
     
     
         3 . The vector of  claim 1  wherein the prokaryotic cells are bacterial cells.  
     
     
         4 . The vector of  claim 1  wherein the polypeptide is a mammalian polypeptide.  
     
     
         5 . The vector of  claim 1  wherein the non-lambda promoter is a trp or alkaline phosphatase promoter or both.  
     
     
         6 . A process for producing a heterologous polypeptide in prokaryotic host cells comprising: 
 (a) culturing the host cells, which comprise (1) anti-termination nucleic acid that inhibits intragenic transcription termination with a non-lambda promoter therefor, and (2) RNA encoding the polypeptide with a non-lambda promoter therefor, wherein an RNA recognition site for binding anti-termination protein produced from the nucleic acid is located 5′ of the RNA encoding the polypeptide, and wherein the anti-termination nucleic acid is expressed at the time of expression of the RNA; and    (b) recovering the heterologous polypeptide from the cells or from cell culture medium.    
     
     
         7 . The process of  claim 6  wherein the heterologous polypeptide is a eukaryotic polypeptide.  
     
     
         8 . The process of  claim 7  wherein the heterologous polypeptide is a mammalian polypeptide.  
     
     
         9 . The process of  claim 8  wherein the mammalian polypeptide is a human polypeptide.  
     
     
         10 . The process of  claim 9  wherein the human polypeptide is thrombopoietin (TPO) or fibroblast growth factor-5 (FGF-5).  
     
     
         11 . The process of  claim 6  wherein the non-lambda promoter is a trp or alkaline phosphatase promoter or both.  
     
     
         12 . The process of  claim 6  wherein the RNA and anti-termination nucleic acid comprise a polycistronic genetic unit comprising a first cistron encoding the heterologous polypeptide and a second cistron downstream from the first cistron that is the anti-termination nucleic acid with a single promoter that controls transcription of said polycistronic genetic unit.  
     
     
         13 . The process of  claim 6  wherein the RNA and anti-termination nucleic acid are expressed under separate promoters.  
     
     
         14 . The process of  claim 6  wherein the prokaryotic cells are bacterial cells.  
     
     
         15 . The process of  claim 6  wherein the polypeptide is recovered from the cytoplasm or periplasm of the cells.  
     
     
         16 . The process of  claim 6  wherein the polypeptide is recovered from the cell culture medium.  
     
     
         17 . The process of  claim 6  wherein the anti-termination nucleic acid is a bacteriophage N or Q gene.  
     
     
         18 . The process of  claim 17  wherein the anti-termination nucleic acid is a lambda N gene.  
     
     
         19 . The process of  claim 18  wherein the RNA recognition site is a nut site.  
     
     
         20 . The process of  claim 19  wherein the nut site is lambda nutL, nutR, Box B, mutant nut, or nut from a lambdoid phage other than lambda phage.  
     
     
         21 . The process of  claim 6  wherein the host cells further comprise nucleic acid encoding a GreA or GreB protein with a promoter therefor.  
     
     
         22 . The process of  claim 21  wherein nucleic acid encoding GreB is expressed.  
     
     
         23 . A vector comprising nucleic acid encoding GreA or GreB protein, nucleic acid encoding a polypeptide heterologous to prokaryotic cells, and one or more promoters for the nucleic acids.  
     
     
         24 . The vector of  claim 23  wherein the nucleic acid encodes GreB.  
     
     
         25 . The vector of  claim 23  wherein the prokaryotic cells are bacterial cells.  
     
     
         26 . The vector of  claim 23  wherein the polypeptide is a mammalian polypeptide.  
     
     
         27 . A process for producing a heterologous polypeptide in prokaryotic host cells comprising: 
 (a) culturing the host cells, which comprise nucleic acid encoding GreA or GreB protein, nucleic acid encoding the heterologous polypeptide, and one or more promoters for the nucleic acids; and    (b) recovering the heterologous polypeptide from the cells or from cell culture medium.    
     
     
         28 . The process of  claim 27  wherein nucleic acid encoding GreB protein is expressed.  
     
     
         29 . The-process of  claim 27  wherein the cells are bacterial cells.  
     
     
         30 . The process of  claim 27  wherein the heterologous polypeptide is a mammalian polypeptide.  
     
     
         31 . The process of  claim 27  wherein the mammalian polypeptide is a human polypeptide.  
     
     
         32 . The process of  claim 31  wherein the human polypeptide is thrombopoietin (TPO) or fibroblast growth factor-5 (FGF-5).  
     
     
         33 . The process of  claim 27  wherein the promoter is a trp or alkaline phosphatase promoter or both.  
     
     
         34 . The process of  claim 27  wherein the polypeptide is recovered from the cytoplasm or periplasm of the cells.  
     
     
         35 . The process of  claim 27  wherein the polypeptide is recovered from the cell culture medium.