The Soluble Expression Strategy In E.coli And Cell-free Protein Biosynthesis System

The purpose of the present work is to increase the expression level of soluble protein both in E. coli and in cell-free protein biosynthesis system. We choose hIGF-1 as our target protein to increase the soluble productivity in E. coli, and then we try to enhance the soluble expression level of eGFP in E. coli cell-free protein biosynthesis system which is riched in chaperones.Firstly, hIGF-1 gene was cloned by RT-PCR from human liver tumor tissues and the expression vector pET32-hIGF-1 was constructed to express hIGF-1 fusion protein. Then the expression levels of TrxA-hIGF-1 were compared by transforming the plasmid (pET32-hIGF-l) into three different E. coli hosts (BL21 (DE3), Rosetta (DE3) and Rosetta-gami (DE3)). The highest productivity of soluble hIGF-1 fusion protein was achieved in E. coli Rosetta-gami (DE3). By using this E. coli, the effects of different expression conditions were investigated systematically to improve the expression level. Under the optimized conditions, a high percent of the target fusion protein was expressed as soluble form with the volumetric productivity of soluble fusion protein reached up to 2.06 g/L. After cell disruption, separation, and cleaved by enterokinase, the concentration of mature hIGF-1 reached up to 0.42 g/L in the mixture.As to E. coli cell-free protein biosynthesis system, we transformed recombinant plasmid with chaperones into E. coli to prepare chaperone-riched extract and used this kind of extract to construct cell-free protein biosynthesis system. By using this chaperone-riched cell-free protein biosynthesis system, the soluble expression level of eGFP was enhanced rapidly.The present work provides a novel approach to increase the productivity of soluble protein both in E.coli and in E. coli cell-free protein biosynthesis system.