Molecular chaperones are folding modulators that play a pivotal role in conformational quality control of proteome and assist proper protein folding upon translation. In this study, the effects of cytoplasmic chaperones on the production of antiTNF-α Fab antibody in Escherichia coli were investigated. To increase the solubility of anti-TNF-α Fab, different chaperone plasmids including pG-KJE8 (GroELS and DnaKJE), pGro7 (GroELS), pKJE7 (DnaKJE), pG-Tf2 (GroELS and TF) and pTf16 (TF) co-expressed in BL21 cells containing pET-22b-Fab construct were used. The solubility of recombinant antiTNF-α Fab was analyzed by SDS-PAGE. The reactivity of the recombinant protein was tested by ELISA and dot blot assays. SDS-PA... More
Molecular chaperones are folding modulators that play a pivotal role in conformational quality control of proteome and assist proper protein folding upon translation. In this study, the effects of cytoplasmic chaperones on the production of antiTNF-α Fab antibody in Escherichia coli were investigated. To increase the solubility of anti-TNF-α Fab, different chaperone plasmids including pG-KJE8 (GroELS and DnaKJE), pGro7 (GroELS), pKJE7 (DnaKJE), pG-Tf2 (GroELS and TF) and pTf16 (TF) co-expressed in BL21 cells containing pET-22b-Fab construct were used. The solubility of recombinant antiTNF-α Fab was analyzed by SDS-PAGE. The reactivity of the recombinant protein was tested by ELISA and dot blot assays. SDS-PAGE analysis of the expressed Fab revealed that chaperone coexpression increased the solubility of the Fab antibody. Among the chaperone sets co expressed with Fab construct, the dnaK/dnaJ/grpE chaperones showed the highest effect on soluble expression with producing about 23.2% soluble Fab antibody. ELISA test results showed, the pKJE7 chaperone led to an approximately sevenfold increase in the activity of produced Fab fragment. Dot blot test results revealed reactivity of recombinant Fab to both transmembrane and soluble form of TNF-α. Our results demonstrated the significant advantage of molecular chaperones in improvement of the soluble expression of Fab antibody in E. coli.
