Methods for cell free protein expression of mature polypeptides derived from zymogens and proproteins

Abstract

High throughput cell free protein synthesis methods and systems are provided for expression of active, mature enzymes and proteins derived from zymogens and proproteins. Zymogens and proproteins are expressed in their mature, active form in a cell free expression system, without cleavage of a pro-sequence to produce the active polypeptide.

We claim: 
     
         1 . A method for expressing a mature, active form of a zymogen or a proprotein, said method comprising: expressing a DNA template that comprises a DNA sequence that encodes a mature polypeptide sequence of a zymogen or proprotein in a cell free expression system that is capable of in vitro transcription from the DNA template and in vitro translation,
 wherein expression in the cell free expression system produces the mature enzyme or protein polypeptide, and   wherein the method does not comprise post-translational processing or cleavage of a pro-sequence from the zymogen or proprotein to produce the mature polypeptide.   
     
     
         2 . The method according to  claim 1 , wherein the cell free expression system further comprises:
 (a) an energy mix that comprises one or more of: polysaccharides, rNTPs, tRNA, CoA, NAD, cAMP, folinic acid, spermidine, and 3-PGA; and   (b) amino acids.   
     
     
         3 . The method according to  claim 1 , wherein the cell free expression system comprises a cell free extract of eukaryotic or prokaryotic cells. 
     
     
         4 . The method of  claim 3 , wherein the cell free expression system comprises a cell free extract of bacterial cells. 
     
     
         5 . The method according to  claim 4 , wherein the bacterial cells comprise  E. coli  cells. 
     
     
         6 . The method according to  claim 1 , wherein the mature polypeptide is expressed as a discrete polypeptide without a pro-sequence. 
     
     
         7 . The method according to  claim 1 , wherein the mature polypeptide is expressed in the presence of a polypeptide pro-sequence for the zymogen or proprotein. 
     
     
         8 . The method according to  claim 7 , wherein a DNA sequence that encodes the pro-sequence and the DNA sequence that encodes the mature polypeptide sequence of the zymogen or proprotein are expressed as discrete polypeptide sequences from the same DNA template. 
     
     
         9 . The method according to  claim 7 , wherein the DNA sequence that encodes the mature polypeptide is expressed from a first DNA template, and the DNA sequence that encodes the pro-sequence is expressed from a separate second DNA template. 
     
     
         10 . The method according to  claim 7 , wherein the pro-sequence is synthesized chemically and added to the cell free expression system prior to, during, or after expression of the mature polypeptide. 
     
     
         11 . The method according to any of  claims 1 - 10 , comprising expressing a mature transglutaminase enzyme (ED 2.3.2.13) in an active, mature form. 
     
     
         12 . The method according to  claim 11 , wherein the transglutaminase enzyme is the transglutaminase from  Streptomyces mobaraensis  (SEQ ID NO:7) or a variant thereof. 
     
     
         13 . The method according to  claim 1 , wherein the DNA template that encodes the mature polypeptide sequence of a zymogen or proprotein is an expression vector or a linear DNA fragment. 
     
     
         14 . The method according to  claim 8 , wherein the DNA template that encodes the mature polypeptide sequence and the pro-sequence polypeptide of a zymogen or proprotein is an expression vector or a linear DNA fragment. 
     
     
         15 . The method according to  claim 9 , wherein the first DNA template and the second DNA template are separate expression vectors or linear DNA fragments. 
     
     
         16 . The method according to any of  claims 13 - 15 , wherein the expression vector is a plasmid derived from pBR322, a pUC vector, or a pET vector. 
     
     
         17 . The method according to any of  claims 13 - 15 , wherein the linear DNA fragment is produced by de novo synthesis or by amplification of a template nucleic acid sequence that encodes the mature polypeptide. 
     
     
         18 . The method of  claim 17 , wherein the amplification comprises polymerase chain reaction (PCR). 
     
     
         19 . The method of  claim 17 , wherein production of the linear DNA fragment comprises a purification step. 
     
     
         20 . The method of  claim 19 , wherein purification of the linear DNA fragment is performed in the presence of a nuclease inhibitor. 
     
     
         21 . The method of  claim 20 , wherein the nuclease inhibitor comprises GamS. 
     
     
         22 . The method of  claim 1 , wherein the cell free expression system further comprises a reversible inhibitor of an activity of the mature zymogen or proprotein. 
     
     
         23 . The method of  claim 1 , wherein the mature polypeptide sequence of the zymogen or proprotein comprises one or more mutation in comparison to the wild type sequence of the zymogen or proprotein. 
     
     
         24 . The method according to  claim 23 , wherein the method comprises:
 introducing one or more mutation into the DNA sequence that encodes the mature polypeptide sequence of a zymogen or proprotein, wherein the one or more mutation results in a variant mature polypeptide sequence; and   expressing the DNA template in the cell free expression system, thereby producing the variant mature polypeptide.   
     
     
         25 . A mature, active polypeptide of a zymogen or proprotein, produced according to the method of any of  claims 1 - 24 . 
     
     
         26 . A cell free expression system for expression of a mature, active form of a zymogen or a proprotein, comprising:
 (a) a nucleic acid template that comprises a DNA sequence that encodes a mature polypeptide sequence of a zymogen or proprotein; and   (b) a cell free extract that is capable of in vitro transcription from the DNA template and in vitro translation to produce the mature polypeptide.   
     
     
         27 . The cell free expression system according to  claim 26 , further comprising:
 (c) an energy mix that comprises one or more of: polysaccharides, rNTPs, tRNA, CoA, NAD, cAMP, folinic acid, spermidine, and 3-PGA; and   (d) amino acids.   
     
     
         28 . The cell free expression system according to  claim 26 , wherein the cell free extract comprises an extract of  E. coli  cells. 
     
     
         29 . The cell free expression system according to  claim 26 , wherein the DNA template encodes a mature sequence of a transglutaminase enzyme (ED 2.3.2.13). 
     
     
         30 . The cell free expression system of  claim 29 , wherein the transglutaminase enzyme is the transglutaminase from  Streptomyces mobaraensis  (SEQ ID NO:7) or a variant thereof. 
     
     
         31 . The cell free expression system according to any of  claims 26 - 30 , further comprising a DNA template comprises a DNA sequence that encodes a pro-sequence for the zymogen or proprotein. 
     
     
         32 . The cell free expression system of  claim 31 , wherein the DNA sequence that encodes the mature polypeptide and the DNA sequence that encodes the pro-sequence of a zymogen or proprotein are encoded as discrete sequences on the same DNA template. 
     
     
         33 . The cell free expression system of  claim 31 , wherein the DNA sequence that encodes the mature polypeptide is encoded on a first DNA template and the DNA sequence that encodes the pro-sequence is encoded on separate second DNA template for the zymogen or proprotein. 
     
     
         34 . The cell free expression system according to any of  claims 26 - 30 , further comprising a chemically synthesized polypeptide pro-sequence for the zymogen or proprotein, which is added to the cell free expression system prior to, during, or after production of the mature polypeptide. 
     
     
         35 . The cell free expression system according to any of  claims 26 - 30 , further comprising a reversible inhibitor of an activity of the mature enzyme or protein. 
     
     
         36 . The cell free expression system of any of  claims 26 - 30 , wherein the mature polypeptide sequence of the zymogen or proprotein is a variant that comprises one or more mutation in comparison to the wild type sequence of the zymogen or proprotein. 
     
     
         37 . An expression vector or linear DNA fragment that comprises a DNA sequence that encodes a mature polypeptide sequence of a zymogen or proprotein and a DNA sequence that encodes a pro-sequence for the zymogen or proprotein, encoded as discrete sequences and separated by a stop codon, a spacer region, a ribosome binding site, and a second start codon. 
     
     
         38 . A composition comprising:
 (a) a first expression vector or linear DNA fragment that comprises a DNA sequence that encodes a mature polypeptide for a zymogen or proprotein; and   (b) a second expression vector or linear DNA fragment that comprises a DNA sequence that encodes a pro-sequence for the zymogen or proprotein.   
     
     
         39 . A method for high-throughput engineering of variants of a zymogen or proprotein, comprising: (i) introducing one or more mutation into a DNA sequence that encodes a mature polypeptide of a zymogen or proprotein, wherein the one or more mutation results in a change in one or more amino acid in the polypeptide sequence, thereby producing a DNA sequence that encodes a variant of the mature polypeptide; and (ii) expressing the DNA sequence in a cell free expression system according to any of  claims 23 - 27 , thereby producing a variant mature polypeptide. 
     
     
         40 . The method according to  claim 37 , wherein the variant DNA sequence is produced by site-directed mutagenesis or de novo DNA synthesis. 
     
     
         41 . The method according to  claim 39 , comprising producing a plurality of different DNA sequences that encode different variants of the mature polypeptide for a zymogen or proprotein, and expressing each of the different DNA sequences in individual cell free expression systems, thereby producing a plurality of different variant mature polypeptides. 
     
     
         42 . The method according to  claim 41 , further comprising assessing one or more property of the variant mature polypeptides, in comparison to the mature form of the zymogen or proprotein from which the variants are derived. 
     
     
         43 . The method according to  claim 42 , wherein the property comprises enzymatic activity and/or stability.