Temperature regulated promoters from schizosaccharomyces pombe for expression of proteins

Abstract

The present invention relates to novel temperature promoters and set of expression vectors isolated from Schizosaccharomyces pombe . The vectors so developed can be used for regulated expression of proteins, both homologous and heterologous, very efficiently and economically.

1 . A process of isolating novel temperature regulated promoters from  Schizosaccharomyces pombe  said process comprising the steps of:
 (a) constructing a partial genomic DNA library with restriction enzyme Sau3AI, to obtain partial genomic DNA sequences in the range of about 100 bp to 2000 bp,   (b) ligating the genomic DNA library sequences of step (a) with vector pGFP without a promoter   (c) transforming the vector of step (b) to  S. pombe  strain,   (d) screening of  S. pombe  strain containing the promoter library,   (e) isolating and identifying two clones of (step d) by stimulating GFP expression,   (f) using the clones obtained in step (e) to check repress or express of GFP expression by temperature shift,   (g) sequencing the genomic DNA fragments of (f) as new promoter elements having SEQ ID No. 1 and SEQ ID No.2, designating the promoters as nmt-185 and nmt-146, useful as promoters, and   (h) cloning the said promoter elements into the novel vectors having Accession nos. MTCC 5106 and 5107 respectively.   
     
     
         2 . A process as claimed in  claim 1 , wherein the step (f) the temperature shifts are 25° C. and 37° C. 
     
     
         3 . A process as claimed in  claim 1 , wherein the promoters have been isolated from  Schizosaccharomyces pombe.    
     
     
         4 . A process as claimed in  claim 1 , wherein the sequence of the said promoter element nmt-185 and nmt-146 is identical or more than 80% homologous to the sequence of nmt1. 
     
     
         5 . A process as claimed in  claim 1 , wherein the promoter element nmt-185 and nmt-146 are repressed in the temperature range of about 33° to 37° C. 
     
     
         6 . A process as claimed in  claim 1 , wherein the promoter element nmt-185 and nmt-147 are expressed in the temperature range of about 22° to 28° C. 
     
     
         7 . A process as claimed in  claim 1 , wherein the promoter element nmt-185 is about 185 bases long. 
     
     
         8 . A process as claimed in  claim 1 , wherein the promoter element nmt-146 is only 146 bases long. 
     
     
         9 . A process as claimed in  claim 1 , wherein the promoter elements nmt-186 and nmt-145 can express or repress the genes GFP, Streptokinase, β-galactosidase and cdc18 gene. 
     
     
         10 . A process as claimed in  claim 1 , wherein GFP expression of said promoters is about 95% within 3 hrs. 
     
     
         11 . A process as claimed in  claim 10 , wherein GFP expression of said promoters is about 91.4% within 3 hrs. 
     
     
         12 . A process as claimed in  claim 1 , wherein said promoters have β-galactosidase activity of about 150±20 units within 3 hrs of induction. 
     
     
         13 . A process as claimed in  claim 12 , wherein said promoters have β-galactosidase activity of about 124.3±20 units within 3 hrs of induction. 
     
     
         14 . A process as claimed in  claim 1 , wherein said promoters have maximum specific activity of about 900 I.U/mg in 3 hrs. 
     
     
         15 . A process as claimed in  claim 38 , wherein said promoters have maximum specific activity of about 870±16 I.U/mg in 3 hrs. 
     
     
         16 . A process as claimed in  claim 1 , wherein said promoters enhance expression of cdc-18 gene within 3 hrs of induction. 
     
     
         17 . A process as claimed in  claim 1 , wherein said promoters give lower leaky expression of proteins. 
     
     
         18 . A process as claimed in  claim 1 , wherein said promoters are not deleterious to the cell viability. 
     
     
         19 . A process as claimed in  claim 1 , wherein said promoters reduce the level of proteolytic degradation. 
     
     
         20 . A process of preparing novel expression vectors based temperature regulated novel promoter elements isolated from  Schizosaccharomyces pombe  said process comprising steps of:
 (a) constructing a partial genomic DNA library with restriction enzyme Sau3AI, to obtain partial genomic DNA sequences in the range of about 100 bp to 2000 bp,   (b) ligating the genomic DNA library sequences of step (a) with vector pGFP without a promoter   (c) transforming the vector of step (b) to  S. pombe  strain,   (d) screening of  S. pombe  strain containing the promoter library,   (e) isolating and identifying two clones of (step d) by stimulating GFP expression,   (f) using the clones obtained in step (e) to check repress or express of GFP expression by temperature shift,   (g) sequencing the genomic DNA fragments of (f) as new promoter elements of 185 bases having SEQ ID NO:1 and 146 bases having SEQ ID NO:2, designated as nmt-185 and nmt-146 respectively, and   (h) cloning the said promoter elements into the novel vectors having Accession vector nos. MTCC 5106 and 5107 respectively.   
     
     
         21 . A process as claimed in  claim 20 , wherein the step (f) the temperature shifts are 25° C. and 37° C. 
     
     
         22 . A process as claimed in  claim 20 , wherein the promoters have been isolated from  Schizosaccharomyces pombe.    
     
     
         23 . A process as claimed in  claim 20 , wherein the sequence of the said promoter element nmt-185 and nmt-146 is identical or more than 80% homologous to the sequence of nmt1. 
     
     
         24 . A process as claimed in  claim 20 , wherein the promoter element nmt-185 and nmt-146 are repressed in the temperature range of about 33° to 37° C. 
     
     
         25 . A process as claimed in  claim 20 , wherein the promoter element nmt-185 and nmt-147 are expressed in the temperature range of about 22° to 28° C. 
     
     
         26 . A process as claimed in  claim 20 , wherein the promoter element nmt-185 is about 185 bases long. 
     
     
         27 . A process as claimed in  claim 20 , wherein the promoter element nmt-146 is only 146 bases long. 
     
     
         28 . A process as claimed in  claim 20 , wherein the promoter elements nmt-186 and nmt-145 can express or repress the genes GFP, Streptokinase, β-galactosidase and cdc18 gene. 
     
     
         29 . A process as claimed in  claim 20 , wherein said vectors have GFP activity of about 95% within 3 hrs. 
     
     
         30 . A process as claimed in  claim 29 , wherein said vectors have GFP activity of about 91.4% within 3 hrs. 
     
     
         31 . A process as claimed in  claim 20 , wherein said vectors have β-galactosidase activity of about 150±20 units within 3 hrs of induction. 
     
     
         32 . A process as claimed in  claim 31 , wherein said vectors have β-galactosidase activity of about 124.3±20 units within 3 hrs of induction. 
     
     
         33 . A process as claimed in  claim 20 , wherein said vectors have maximum specific activity of about 900 I.U/mg in 3 hrs. 
     
     
         34 . A process as claimed in  claim 33 , wherein said vectors have maximum specific activity of about 870±16 I.U/mg in 3 hrs. 
     
     
         35 . A process as claimed in  claim 20 , process as claimed in  claim 24 , wherein said vectors enhance expression of cdc-18 gene within 3 hrs of induction. 
     
     
         36 . A process as claimed in  claim 35 , wherein said vectors give lower leaky expression of proteins. 
     
     
         37 . A process as claimed in  claim 20 , wherein said vectors are not deleterious to the cell viability. 
     
     
         38 . A process as claimed in  claim 20 , wherein said vectors reduce the level of proteolytic degradation.