Independently Inducible System of Gene Expression

Abstract

The present invention is directed to the improved methods for the temporal induction of proteins using the condensed single protein production (cSPP) system.

1 . A vector comprising:
 a. a cspA cold shock promoter;   b. a tetR gene;   c. a tet operator; and   d. a gene encoding a target protein under the control of the tet operator.   
     
     
         2 . The vector of  claim 1 , further comprising a sequence encoding at least one fusion tag. 
     
     
         3 . The vector of  claim 2 , wherein the fusion tag increases translational efficiency, aids in purification of the target protein, or both. 
     
     
         4 . The vector of  claim 2 , wherein the fusion tag is a translational enhancing element or a His 6  tag. 
     
     
         5 . The vector of  claim 2 , wherein the fusion tag sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 12. 
     
     
         6 . The vector of  claim 4 , wherein the fusion tag comprises a His 6  tag, further comprising a Factor Xa protease cleavage site or a TEV protease cleavage site. 
     
     
         7 . The vector of  claim 1 , wherein the target protein is capable of being induced by tetracycline or an analog thereof. 
     
     
         8 . The vector of  claim 1 , wherein the target protein is ACA-less. 
     
     
         9 . The vector of  claim 1 , further comprising a gene encoding an OmpA signal peptide. 
     
     
         10 . The vector of  claim 1 , wherein the gene encoding the target protein is sub-cloned into the vector between NdeI and BamHI restriction sites. 
     
     
         11 . A cell containing:
 a. a vector comprising a gene encoding a target protein; and   b. a vector comprising a gene encoding an mRNA specific endoribonuclease, wherein the target protein and mRNA-specific endoribonuclease are capable of being induced with different substances.   
     
     
         12 . The cell of  claim 11 , wherein the mRNAs associated with the expression of the target protein are do not contain the sequence at which the mRNA-specific endoribonuclease cleaves. 
     
     
         13 . The cell of  claim 11 , wherein the mRNA-specific endoribonuclease is MazF. 
     
     
         14 . The cell of  claim 11 , wherein the mRNA-specific endoribonuclease is capable of being induced by IPTG. 
     
     
         15 . The cell of  claim 11 , wherein the target protein is capable of being induced with tetracycline or an analog thereof. 
     
     
         16 . The cell of  claim 11 , wherein the mRNA-specific endoribonuclease is capable of being induced by 3-β-indoleacrylic acid (IAA), L-arabinose, or L-rhamnose. 
     
     
         17 . The cell of  claim 11 , wherein the target protein is capable of being induced by 3-β-indoleacrylic acid (IAA), L-arabinose, or L-rhamnose. 
     
     
         18 . A method of labeling a target protein comprising:
 a. contacting a culture of cells of  claim 11  with a substance capable of inducing the mRNA-specific endoribonuclease; and   b. contacting a culture of the cells of  claim 11  with an isotope-enriched medium comprising a substance capable of inducing the target protein.   
     
     
         19 . The method of  claim 18 , further comprising condensing the culture to a desired culture volume. 
     
     
         20 . The method of  claim 19 , wherein the substance capable of inducing the target protein is anhydrotetracycline, and wherein the volume of anhydrotetracycline present is between about 0.1 μg/mL and about 0.2 μg/mL multiplied by the degree of condensation. 
     
     
         21 . The method of  claim 19 , wherein the substance capable of inducing the target protein is anhydrotetracycline, and wherein the volume of anhydrotetracycline present is about 0.15 μg/mL. 
     
     
         22 . The method of  claim 18 , wherein at least 80% of the target protein is labeled. 
     
     
         23 . The method of  claim 18 , wherein at least 85% of the target protein is labeled. 
     
     
         24 . The method of  claim 18 , wherein at least 90% of the target protein is labeled. 
     
     
         25 . The method of  claim 18 , wherein at least 95% of the target protein is labeled. 
     
     
         26 . The method of  claim 18 , wherein the substance capable of inducing the target protein is tetracycline or an analog thereof. 
     
     
         27 . The method of  claim 18 , wherein the substance capable of inducing the target protein is anhydrotetracycline. 
     
     
         28 . The method of  claim 18 , wherein the substance capable of inducing the target protein is 3-β-indoleacrylic acid (IAA), L-arabinose, or L-rhamnose. 
     
     
         29 . The method of  claim 18 , wherein the substance capable of inducing the mRNA-specific endoribonuclease is 3-β-indoleacrylic acid (IAA), L-arabinose, or L-rhamnose. 
     
     
         30 . A protein labeled by the method of  claim 18 . 
     
     
         31 . The protein of  claim 30 , comprising at least one fusion tag. 
     
     
         32 . The protein of  claim 31 , wherein the fusion tag increases translational efficiency, aids in purification of the target protein, or both. 
     
     
         33 . The protein of  claim 31 , wherein the fusion tag is selected from the group consisting of a translational enhancing element and a His 6  tag.