Cellular engineering strategies for soluble and secretory recombinant human insulin production in Pseudomonas...

One of the main challenges in expressing recombinant human insulin is the formation of inclusion bodies. In our previous study, we successfully expressed insulin in Pseudomonas fluorescens. We observed that lowering the post-induction temperature enhanced the soluble fraction; however, it resulted in lower protein titer. In this study, the individual and synergistic effects of multiple chaperones and signal peptides on soluble protein synthesis are analyzed. A plasmid with a spectinomycin antibiotic resistance encoding gene was constructed for co-expression of chaperones. The combined effect of Disulphide bond protein A (DsbA) and phosphate binding protein (Pbp) resulted in ∼60 % of the fusion protein in soluble form. A comprehensive study was performed to assess the influence of the location of the His-tag on the expression and solubility of the protein. A high-copy origin of replication (ori) produced a lower soluble protein titer and caused a significantly longer lag phase. Under optimal concentration of antibiotics and inducer, inoculation percentage and induction OD₆₀₀, the protein and soluble protein fraction titer increased by ∼58 % and ∼27 %, respectively. In a batch bioreactor, a maximum of 234.58 mg/l fusion protein with 71.59 % solubility and around 15 % of the total soluble protein fraction in the culture supernatant was obtained. Circular dichroism analysis revealed that the secondary structure of the purified insulin was comparable to that of the standard insulin. To the best of our knowledge, this is the first study reporting the combinatorial effect of chaperones and signal peptides on the production of human insulin in the soluble form.