CLONING AND SOLUBLE OVER-EXPRESSION OF HUMAN GROWTH HORMONE(TRX-HIS6-HGH) IN ESCHERICHIA COLI

Background and Aim:Human growth hormone is a 22kDa polypeptide which consists of 191 amino acids and two disulfide bonds. This hormone has therapeutic applications. E.coli is the preferred host since hGH has no need to post translational modification. Often the protein over-expression in E.coli is led to inclusion bodies (IBs) formation, this form has some disadvantages such as misfolding of target protein, solubilization and refolding in downstream process. The expression of hGH fused with Trx, NusA, or FH8 tags make it soluble and folded in E. coli. In this study, Trx-His6-hGH was expressed in soluble form in E.coli Rosettagami strain.Methods:The designed gene cassette, from 5 to 3, consists of Trx, His6, enterokinase and hGH respectively. The cassette is first cloned in the pET-32a(+) plasmid and then transferred to E.coli DH5 and afterward transferred to the E.coli Rosettagami. The expression of hGH was performed in Luria Bertani(LB) and Terrific Broth(TB) in 25℃ in shaking flask. Results were analyzed by SDS-PAGE and confirmed with western blot.Results:SDS-PAGE analysis showed that 58% and 55.5% of total protein expressed belongs to Trx-His6-hGH in LB and TB respectively. As the amount of biomass is important for next downstream processes, the results from TB was accepted. Of 55.5% of hGH expressed 33.7% and 18.8% expressed in soluble and IBs form respectively.Conclusion:According to the results of this study, over-expression of soluble hGH in cytoplasm of E.coli can be achieved appropriately when fused with Trx tag.

کلیدواژه ها:

Recombinant Human Growth Hormone ، rhGH ، Trx-hGH ، Cytoplasmic over-expression of rhGH ، Cloning of Trx-His6-hGH ، Trx-His6-hGH