Abstract
Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.
Highlights
Engineered strains of E. coli have been well-documented as ideal and often first- choice expression systems when fast and economical production of recombinant proteins is desired.[1,2,3] This system has several advantages over other expression systems including well-characterized genetic structure, easy cultivation in inexpensive culture media, and rapid biomass accumulation.[4]
The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies
Despite advantages mentioned for E. coli, several limitations including formation of inclusion bodies (IBs) in case of aggregation- prone proteins, system inability in yielding proper soluble proteins of large- size (>60 kD), impotency of the system in producing glycosylationneeded proteins, and disturbance of correct folding imposed by unfavorable disulfide bonds formation, are restrictive factors in using this host for recombinant protein production.[6,7,8]
Summary
Engineered strains of E. coli have been well-documented as ideal and often first- choice expression systems when fast and economical production of recombinant proteins is desired.[1,2,3] This system has several advantages over other expression systems including well-characterized genetic structure, easy cultivation in inexpensive culture media, and rapid biomass accumulation.[4]. In our study the effects of plasmid chaperones pGro[7] containing GroES-GroEL chaperone team, pG-Tf2 containing GroES- GroEL- tig chaperone team and pTf16 containing tig chaperone on soluble expression of hscFv were investigated
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