DiscussionFaiza 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.
SRP mediated DsbA signal sequence (DsbAss) at the N terminus of ovine growth hormone gene (oGH) in a plasmid construct under T7 promoter resulted in biologically active oGH in the inner membrane of E. coli. Optimum expression of oGH (ovine growth hormone) with DsbAss was achieved in 0.6M mannitol, 50μM ZnCl2, 50mM glycylglycine supplemented TB medium and induction with 50μM IPTG in the early logarithmic phase to a final OD600 ~3.10 at 25ºC in shake flask at 150rpm. The 90% pure soluble oGH was achieved in high yield of 443mg/l from the inner membrane of E. coli by simple ultracentrifugation. MALDI TOF, Western blot and HeLa cell line proliferation proved its authenticity. Further investigation on the effect of amino acid substitutions in three regions of DsbAss to target recombinant oGH via the SRP pathway showed the importance of Ala13 in the H domain of DsbAss for its binding with Ffh component of SRP.