Cytoplasmic soluble Lispro insulin production in Escherichia coli, product yield optimization and physiochemical characterization

Lispro is an insulin analog with an accelerated action profile suitable for post-prandial injections and glycemic control in diabetes. Insulin production in Escherichia coli leads to inclusion body formation due to its reducing inner environment. This conventional platform requires refolding procedures with relatively low recovery resulting in product loss and increased production time. We introduced a novel approach for high yield cytoplasmic soluble expression of Lispro insulin in E. coli . Presence of the SUMO fusion tag and being expressed in SHuffle T7 improved the solubility of Lispro precursor to10 mg/L. Product yield increased to approximately 5-fold after the optimization procedure. The scalability of the optimized condition was confirmed in a fed-batch fermentor that resulted in high cell density and high yield soluble expression (540 mg/L). 25 mg/L bioactive Lispro insulin was generated and purified. The final product was identical to the commercial analog based on physiochemical characterization analyses. • Insulin production in E. coli is highly affected by Inclusion body formation. • Inclusion bodies were avoided by the use of SUMO fusion tag and SHuffle T7 strain. • RSM-based optimization increased the yield of soluble Lispro proinsulin by 5-fold. • Large scale fermentation yielded 540 mg/L soluble Lispro proinsulin. • The soluble proinsulin was converted to bioactive Lispro (25 mg/L final product).