The Construction Of Escherichia Coli Strains With Various Copies Of GroELS For Soluble Production Of Recombinant Proteins By Tn5 Transposase Method

The overproduction of recombinant proteins in Escherichia coli often generate inclusion bodies when the proteins cannot fold properly.Co-overproduction of molecular chaperones,such as GroELS?DnaK-DnaJ-GrpE and Trigger factor,can facilitate protein folding and enhance the production of soluble and active proteins.Several plasmids with molecular chaperones have been constructed to deal with this problem.However,the strategy has some disadvantages,including the tendency of plasmid loss and the use of antibiotics for maintaining the plasmids.It is also difficult to control the production of chaperon proteins from plasmids;however,the proper concentrations of the chaperones are important as too many chaperones may lead to enhanced proteolysis and reduced production of recombinant proteins.Here,a Tn5 transporase method was adapted for the integration of genes into the genome one copy at a time.The E.coli BL21(DE3)strains with 1-4 copies of groELS were obtained,and the strains was tested for better production of soluble proteins.1.The adaptation of a Tn5 transposase method for the multiple integration of genesThe expression vector pET28a::tnpA was constructed for transposase production.The His-tagged transposase was purified with a modified Ni-agarose method,in which NaCl concentration was increased to 1 M to wash off contaminated proteins.The method was further optimized with the purified transposase.A Kan selection marker gene flanked by FRT sites was added behind groELS.The kan gene can be removed thro?gh FLP site-specific recombination.This method could be used for the integration of one copy or multi-copies of a gene.The method may be further used for synthetic biology and metabolic engineering.2.The construction of engineered bacteria with various copies of groELS genesThe groELS genes were successfully constructed under the control of phage T7 promoter and integrated onto E.coli BL21(DE3)chromosome.The process was repeated to obtain four strains with 1 to 4 copies of groELS.To our knowledge,this is the first report for multiple-gene integration by using Tn5 transposase.The integrated groELS did not affect cell growth.Compared with the bacteria that expressed groELS on plasmids,the engineered strains are more useful for industrial applications since it does not need antibiotics to maintain the plasmid.3.The best copy number of groELS for producing soluble recombinant peoteinsThe four strains with increased copies of groELS produced increased amounts of GroELS,proportional to the copies.They were used to test the effect of GroELS concentrations on the optimal production of selected recombinant proteins:a cytoplasmic monooxygenase EmoA from bacterium Chelativorans sp.BNC1,a mitochondrial matrix sulfur dioxygenases ETHE1 from human,and a membrane protein sulfide:quinone oxidoreductase Sqr from Pseudomonas aer?ginosa PAO1,respectively.The E.coli strains with 2-3 copies of GroELS were found to be the best for achieving the most soluble proteins for all three tested recombinant proteins.The results support the hypothesis that the quantity of expressed soluble proteins was affected by the quantity of GroELS.The engineered strains may be useful for the production of recombinant proteins.