Optimized conditions for high-level solubilization and purification of recombinant camel growth hormone in Escherichia coli
In this report, we describe the cloning, over-expression, efficient solubilization, purification and evaluation of bioactivity of camel growth hormone (cGH). The total cellular RNA was extracted from pituitary glands of freshly slaughtered animals and cDNA of cGH was synthesized by a pair of sequence specific primers with a product of 576 base pairs (bps). Amplicons was cloned into T/A cloning vector and positive clones were subjected to sequencing. After sequencing, cDNA was cloned in the prokaryotic expression vector system pET23b+. Conditions for cGH expression were optimized by varying the concentration of isopropyl-L-thio-β D-galactopyronoside (IPTG) and induction time. It was observed that 100 μM concentration of IPTG and 3 h post-induction produced the highest amount of cGH. Expressed GH was sequestered as inclusion bodies (IBs), and was therefore, solubilized using denaturant (urea) and detergents (SDS, CTAB, Tritin X-100, Tween-20). The best solubilization was obtained with 8.5 mM SDS in 100 mM Tris buffer at pH 8.5. The solubilized cGH was purified by gel filtration chromatography using Sephadex G-50 column. The purified protein was refolded by dialysis, analyzed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blot. Further biological activity of purified product was confirmed by efficient growth of rat Nb2 lymphoma cells. This study provided the method for the efficient solubilization of cGH (r-cGH) with comparable bioactivity with commercially available bovine growth hormone (bGH) and could be further used for solubilization of other proteins expressed in prokaryotic system.
Key words: Recombinant growth hormone, somatotropin, cloning, expression, inclusion bodies, solubilization, purification, bioactivity.