Study of the impact of different induction conditions and T7 promoter expression on recombinant IGF-I production in Escherichia coli
چکیده
Introduction: Insulin-like growth factor 1 (IGF-1) is a growth factor that promotes pituitary growth hormone action and thus mediates protein anabolic and linear growth. Mecasermin (human recombinant IGF-1) is used to treat growth failure in children with primary IGF-1 deficiency over the long term. To gain the benefits of recombinant IGF-1 for medicinal purposes, we must be able to overproduce it. However, determining the ideal manufacturing conditions for all proteins is still a difficult task. Aside from temperature and induction conditions, the promoter used, the bacterial strain used, and the solubility of the target protein are all factors that influence total protein production and the amount of soluble protein produced.
Aim: In this thesis project, we aimed to substitute trc promoter with T7 promoter, which is stronger, and determine the effect of culture and induction conditions on recombinant IGF-1 production in Escherichia coli.
Methods: After altering the promoter in the expression cassette, we performed cloning and expression of a synthetic IGF-1 gene in the SHuffle strain of E. coli. The bacteria were grown in SB, TB, 2xYT, SOB, and SOC to find the best culture media. A preliminary test was done to gain insight into the effect of IPTG concentration. The central composite design of response surface methodology was applied to determine the optimum IPTG concentration and post-induction time.
Results: The most amount of biomass and rIGF-1 was observed in bacteria grown in SOB and SOC media. An increase in the IPTG concentrations from 0.4 mM to 0.8 mM and 1.2 mM showed further amounts of expression. In the central composite design, the star points, which were 6h of induction and 1 mM IPTG concentration, demonstrated the highest expression.
Conclusion: In SOC media, the moderate IPTG concentration and post induction time have significant effect on rIGF-1 expression