Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH wa s produced at a cell concentration of 25 g dry cell weight/L. Inclusion bod ies from the cells were isolated and purified to homogeneity. Various buffe rs with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic int eractions were found to be the dominant forces responsible for the formatio n of r-hGH inclusion bodies during its high-level expression in E. coli. Co mplete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion b odies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yie ld of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was pur ified by use of DEAE-Sepharose ion-exchange chromatography and the pure mon omeric r-hGH was finally obtained by using size-exclusion chromatography. T he overall yield of the purified monomeric r-hGH was similar to 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines. (C) 2000 Academic Press.