Persian sturgeon growth hormone elaboration and purification

In this study Escherichia coli DE۳ containing expression vector (pET۲۱a) with cloned Persian sturgeon growth hormone (psGH) gene was grown in ۱۰ mL LB broth on a ۱۵۰ rpm shaker, at the temperature of ۳۷ °C. At the late log phase (determined by OD standard curve) ۱۰۰ ;muL isopropyl ;beta-D-۱-thiogalactopyranoside (IPTG) was added for induction of GH synthesis. Samples were taken every ۲ hours and after bacterial cells lysis crude extracts with recombinant proteins inclusion bodies (IB) were loaded on ۱۵% SDS-PAGE gel. Thenafter staining, comparative concentrations of rpsGH were measured by densitometric scanning of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel  it was more than ۹۰ %. The maximum yield of GH was observed after ۴ hours of growth. To recover active psGH from inclusion bodies we used imidozole to obtain most of the total recombinant protein in the soluble fraction. Purification of ۶xhisN tag recombinant psGH has been performed using affinity chromatography where nickel was bound to an agarose bead by chelation using NTA (nitrilotriacetic acid) beads. The overall yield of the purified monomeric psGH was approximately ۵۰% of the initial IB proteins. The purification manipulations including IB isolation and solubilisation, protein refolding by dialyze and affinity chromatography ensure yields of biologically active psGH up to ۳۰%. This study shows that, the affinity chromatography is a powerful and very specific method for recombinant proteins purification of  psGH.