Discussion

Cost effective production of biologically active recombinant ovine growth hormone via SRP secretory system
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.
faiza_phd@hotmail.com
Back ground
Growth hormone (GH), a protein of 22kD promotes somatic growth and regulates the lipid mobilization in vertebrates. Several groups have reported the expression of GH or its derived fusion protein in E. coli cytoplasm. However, the cytoplasmic production of a protein has certain disadvantages: high level accumulation often leads to insoluble protein aggregates that can be difficult to refold, solubilize and a refolding step is frequently required to obtain the native conformation to form the correct disulfide bonds. Alternative expression systems have been based on the secretion of the protein into the E. coli periplasmic space, which not only allow a greater chance to obtain the protein in a folded and soluble form but lower load of contaminating proteins in the periplasmic fluid makes purification process easier. Most of the soluble recombinant GH so far studied have been translocated to the periplasmic space which accounts for 6% of the cell compartment so resulting in less yield of recombinant protein. Here we report the translocation of roGH to inner membrane( which is 21 % of the cell )in order to achieve high yield of protein.
Aim of the work
The aim of the project was cost effective production of a biologically active recombinant growth hormone in Escherichia coli.
Methods and result
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. In silico structural analysis of oGH binding to GHBp revealed another crucial amino acid, Glu62, present in hGH, bGH and oGH, showed the binding of GHR across the Bovidae group.