Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway
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Expression of recombinant protein that possess disulfide bonds especially Fab antibody fragment in periplasm of E. coli is the most favorable platform. But formation of inclusion bodies and inefficient translocation of desired protein to the periplasm is considerable obstacle threatening the advantages of E. coli expression system. Co-expression with molecular chaperones is remarkable consideration to solve this problem. In this study we evaluated the effect of co-expression of molecular chaperones and optimization condition on efficient periplasmic translocation of Fab antibody fragment. Plasmid pKJE7 was used for co-expression of DnaK/DnaJ/GrpE in BL21 cells and SHuffle strain (constantly expressing DsbC chaperon) were used for evaluation of chaperones effects. Periplasmic fraction was prepared by osmotic shock method and ELISA test were used for activity measurement. The Fab antibody purified by IMAC chromatography. The results indicated that co-expression of anti-TNF-α Fab with DnaK/DnaJ/GrpE in BL21 cells had a marked effect on the yield of periplasmic fraction but DsbC had no effect on periplasmic translocation. The optimal culture condition was found 0.1 mM IPTG concentration and 25 °C temperatures. SDS-PAGE analysis indicated that solubility increased to 42% of total Fab in Bl21 co-expressing DnaKJE. This study reports that co-expression with DnaK/DnaJ/GrpE markedly increases soluble expression of Fab antibody fragment in optimized condition.
KeywordsFab fragment Certulizumab Molecular chaperone Protein disulfide-isomerases
This study was funded by Drug Applied Research Center (Grant No. 94/54), Tabriz University of Medical Science, Tabriz, Iran. The results presented in this work are based on data set of PhD thesis, recorded in Tabriz University of Medical Sciences.
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