Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli.

Abstract

Ovine growth hormone was expressed in Escherichia coli in the form of inclusion bodies using the pQE-30 expression vector. In a simple fed-batch fermentation, 800 mg/L of recombinant ovine growth hormone (r-oGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies were isolated from cells with >95% purity by extensive washing using detergent, and the r-oGH from the purified inclusion bodies was solubilized in 2 M Tris-HCl buffer at pH 12 containing 2 M urea. The r-oGH solubilized in the above conditions exhibited considerable secondary structure as determined by circular dichroism spectra and was immunologically active. Solubilization of the inclusion body protein with retention of native-like secondary structure gave higher yields during refolding. To suppress protein aggregation, refolding was carried out in gel filtration column. Refolding, buffer exchange, and the purification of monomeric r-oGH from aggregated complex was achieved in a single step using gel filtration chromatography. More than 60% of the initial inclusion body protein was refolded into a native-like conformation by the use of this procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS-PAGE, Western blotting, and radio receptor binding assay and found to be similar to native, pituitary-derived, ovine growth hormone.