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Persian sturgeon growth hormone elaboration and purification | ||
| Iranian Journal of Fisheries Sciences | ||
| مقاله 20، دوره 14، شماره 4، دی 2015، صفحه 1053-1060 اصل مقاله (313.54 K) | ||
| شناسه دیجیتال (DOI): 10.22092/ijfs.2018.114503 | ||
| نویسندگان | ||
| E Nasr* ؛ H.G Hovhannisyan؛ M Pourkazemi | ||
| چکیده | ||
| In this study Escherichia coli DE3 containing expression vector (pET21a) with cloned Persian sturgeon growth hormone (psGH) gene was grown in 10 mL LB broth on a 150 rpm shaker, at the temperature of 37 °C. At the late log phase (determined by OD standard curve) 100 &muL isopropyl &beta-D-1-thiogalactopyranoside (IPTG) was added for induction of GH synthesis. Samples were taken every 2 hours and after bacterial cells lysis crude extracts with recombinant proteins inclusion bodies (IB) were loaded on 15% 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 90 %. The maximum yield of GH was observed after 4 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 6xhisN 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 50% 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 30%. This study shows that, the affinity chromatography is a powerful and very specific method for recombinant proteins purification of psGH. | ||
| کلیدواژهها | ||
| Persian sturgeon؛ Growth hormone؛ PURIFICATION؛ Affinity chromatography | ||
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