Discussion

Faiza Gul Durrani1, Roquya Gul1,2,Muhammad Usman Mirza2, M. Waheed Akhter1.
School of Biological Sciences, University of the Punjab, Lahore, Pakistan1.
University of the Lahore,Lahore,Pakistan2.

Abstract

This study involves periplasmic expression of roGH via T 7 promoter system using PET 22b vector. Total RNA was isolated from pituitary gland of local ovine breed (Lohi) by using Guanidium-thiocyanate-chloroform extraction method. cDNA was synthesized by RT-PCR using specific primers. Recombinant proteins were expressed using pET vector in periplasmic space of E. coli BL21DE3. Best result of 22% expressed roGH on SDS-PAGE was observed at 20M (final concentration) IPTG after 4 hrs of fermentation at 370C in LB modified medium with 50M ZnCl2 in BL21DE3 E.coli strain. The optimized freeze thaw method including 25% glycerol with 50M ZnCl2 enhanced the relase of oGH upto 24% in the periplasmic space of E. coli . The oGH thus found was further purified by FPLC and authenticated by Western blot analysis.In silico structural analysis of binding of oGH with GHBP revealed a crucial amino acid Glu62 which is present in hGH, bGH and oGH ,presence of this showed the binding of GHr across the bovidae group.