Design and construction of rh-IGF-I fusion protein expression vector and its cloning in E.Coli

Abstract

Introduction: IGF-I is an anabolic hormone produced by liver cells in response to growth hormone. The production of this hormone altered in some metabolic diseases including Laron syndrome, Liver cirrhosis, aging, cardiovascular and neurological diseases. Therefore, replacement therapy using human recombinant IGF-I protein entitled "Mecasermin" is necessary to rehabilitate the patients. Objectives: Designing of soluble rh-IGF-I expression cassette, and its cloning in E. coli Method: Human IGF-I encoding sequence was attached in-frame to the sequence of different solubilizing proteins using the desired linker. The solubility of the new fusion proteins were analyzed using PROSO II online server; and, the most solubilized fusion protein with lowest amount of the amino-acids was selected. Afterward, for construction of expression cassette, the related sequences of promoter, lac operator, ribosome binding site, and terminator were added to the designed rh-IGF-I fusion protein sequence. The overall designed expression cassette was cloned in the back bone of pET-22b expression vector, and transformed in to the E.Coli Origami. The analogue of lactose "IPTG" was used to induce the expression of rh-IGF-I fusion protein; and, its production was confirmed using SDS-PAGE and subsequent silver staining techniques. Results: As there wasn't any detergent and solubilizing enhancer agent, in opted protein extraction buffer, the existences of the 40 kDa related protein band in Silver-Stained SDS-PAGE analysis of induced and non-induced control samples indicated the soluble rh-IGF-I protein production in E.Coli host. Conclusion: Combined usage of designing appropriate expression cassette, suitable E.Coli strain and optimized induction condition are necessary for soluble recombinant human IGF-I production.