Evaluating the impact of different engineered E. coli strains on Recombinant IGF-I production

Abstract

Introduction: Insulin-like growth factor 1 (IGF-I) is a growth factor that promotes pituitary growth hormone action and thus mediates protein anabolic and linear growth. Mecasermin (human recombinant IGF-I) is used to treat growth failure in children with primary IGF-I deficiency over the long term. To gain the benefits of recombinant IGF-I 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 type, the bacterial strain, and the solubility of the target protein are all factors that influence total protein production.Aim: In this thesis project, we aimed to study the impact of different strains of E. coli on the production of recombinant IGF-I.Methods: After cloning the expression vector into different strains of E. coli, the single colony of each group was cultivated in liquid culture LB media, and IGF-I production was induced using IPTG with a concentration of 0.4 mM for 4h. Then, Real-time PCR was performed to evaluate the production capability of each E. coli strain. In this regard, at first total RNA of each sample was extracted and converted to cDNA. Then, by using IGF-I-specific primers RT-PCR was performed and compared the gene expression between different strains of E. coli.The result of RT-PCR showed that the SHuffle strain had a better expression rate than other E. coli strains. Subsequently, the central composite design of response surface methodology was applied to determine the optimum IPTG concentration and induction time.Result: in the central composite design the SHuffle strain with 4h of induction and 1mM IPTG concentration, demonstrated the highest expression. Conclusion: The moderate IPTG concentration and post-induction time have a significant effect on IGF-I expression.