Production of recombinant human insulin from a promising Pseudomonas fluorescens cell factory and its kinetic modeling.

Insulin intake is recommended for diabetics in addition to a proper diet and lifestyle to maintain adequate blood glucose level. Currently, there is a need for an alternative expression system for insulin production as the current expression systems may not meet the growing demand due to various constraints. Here, we demonstrate the synthesis of human insulin in an unconventional expression system based on Pseudomonas fluorescens, a BSL 1 bacterium. Human insulin was produced in the form of proinsulin fused with fusion protein. Then, the proinsulin fusion protein was purified using Ni-NTA chromatography and converted into human insulin. The physicochemical parameters for producing proinsulin fusion protein are optimized. Glucose and ammonium chloride are determined to be suitable carbon and nitrogen sources, respectively. The validity of insulin and proinsulin fusion protein is assessed using western blot and quantified using ELISA techniques. Up to 145.35 mg/l of the proinsulin fusion protein is achieved at the shake flask level. Further, MALDI-TOF and RP-HPLC analysis of the purified human insulin were observed to be close to the theoretical value and insulin standard, respectively. The expression of the recombinant fusion protein was found to be 214.7 mg/l in a batch bioreactor, a ∼48% enhancement over the shake flask level. Further, kinetic modeling was performed to understand the system regarding growth, substrate utilization and product formation, and to estimate the various kinetic parameters. This study establishes the potential of the P. fluorescens expression system for producing human insulin.