Transcript optimized expression enhancement for high-level production of proteins and protein domains

Abstract

The present invention relates to a system for high-level production of recombinant proteins and protein domains.

1 . A method of preparing an expression vector, wherein the expression vector comprises, in order of position: a first nucleic acid sequence encoding a 5′ untranslated region of an expressed mRNA that comprises a ribosome binding site (RBS); a second nucleic acid sequence encoding a polypeptide tag; and a cloning site, wherein the cloning site enables a target protein coding sequence to be inserted into the vector in-frame with the second nucleic acid sequence to encode a fusion protein comprising the polypeptide tag and the target protein; and wherein the method comprises specifically modifying the nucleic acid sequence encoding (i) the 5′ untranslated region and (ii) the adjacent polypeptide tag to minimize RNA secondary structure both within and/or between these two regions of the mRNA. 
     
     
         2 . The method of  claim 1 , further comprising specifically modifying the second nucleic acid sequence to reduce the presence of rare codons. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the expression vector further comprises a target protein coding sequence inserted into the vector in-frame with the nucleic acid tag sequence to encode a fusion protein comprising the polypeptide tag and the target protein. 
     
     
         6 . The method of  claim 5 , wherein the target protein coding sequence is not modified to minimize RNA secondary structure and/or is not modified to reduce the presence of rare codons. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the second nucleic acid sequence encodes at least one affinity purification tag. 
     
     
         9 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein the second nucleic acid sequence encodes at least one solubility enhancement tag. 
     
     
         14 - 18 . (canceled) 
     
     
         19 . The method of  claim 5 , wherein the target protein coding sequence encodes a transcription factor, a transcription factor domain, an epigenetic regulatory factor, or an epigenetic regulatory factor domain. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 5 , wherein the target protein coding sequence encodes a protein antigen for producing an affinity capture reagent. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . The method of  claim 5 , wherein the expression of the target protein is 1.5 fold greater than the expression of a target protein generated from an expression vector that was not modified as described in  claim 1 . 
     
     
         25 . An expression vector prepared using the method of  claim 1 . 
     
     
         26 . An expression vector comprising, in order of position: a first nucleic acid sequence encoding a 5′ untranslated region of an expressed mRNA that comprises a ribosome binding site (RBS); a second nucleic acid sequence encoding a polypeptide tag; and a cloning site, wherein the cloning site enables a target protein coding sequence to be inserted into the vector in-frame with the second nucleic acid sequence to encode a fusion protein comprising the polypeptide tag and the target protein; and wherein the nucleic acid sequence encoding (i) the 5′ untranslated region and (ii) the adjacent polypeptide tag has been specifically modified to minimize RNA secondary structure both within and/or between these two regions of the mRNA. 
     
     
         27 . The expression vector of  claim 26 , wherein the second nucleic acid sequence has been specifically modified to reduce the presence of rare codons. 
     
     
         28 . The expression vector of any one of  claims 26 - 27 , wherein nucleotides within about the last 100 nucleotides of the first nucleic acid sequence have been modified. 
     
     
         29 . The expression vector of any one of  claims 26 - 28 , wherein nucleotides within about the first 90 nucleotides of the second nucleic acid sequence have been modified. 
     
     
         30 . The expression vector of  claim 26 , further comprising a target protein coding sequence inserted into the vector in-frame with the nucleic acid tag sequence to encode a fusion protein comprising the polypeptide tag and the target protein. 
     
     
         31 . The expression vector of  claim 30 , wherein the target protein coding sequence has not been modified to minimize RNA secondary structure and/or has not been modified to eliminate rare codons. 
     
     
         32 . (canceled) 
     
     
         33 . The expression vector of  claim 26 , wherein the second nucleic acid sequence encodes at least one affinity purification tag. 
     
     
         34 - 37 . (canceled) 
     
     
         38 . The expression vector of  claim 26 , wherein the second nucleic acid sequence encodes at least one solubility enhancement tag. 
     
     
         39 - 43 . (canceled) 
     
     
         44 . The expression vector of  claim 30 , wherein the target protein coding sequence encodes a transcription factor, a transcription factor domain, an epigenetic regulatory factor, or an epigenetic regulatory factor domain. 
     
     
         45 - 48 . (canceled) 
     
     
         49 . The expression vector of  claim 30 , wherein the target protein is expressed at a 1.5-fold higher level than a target protein generated from an expression vector that was not modified as described in  claim 26 . 
     
     
         50 . A host cell comprising the expression vector of  claim 30 . 
     
     
         51 . A method for expressing a target protein in a host cell, comprising culturing the host cell of  claim 50  for a period of time under conditions permitting expression of the target protein. 
     
     
         52 - 54 . (canceled)