Optimizing proinsulin production in E. coli BL21 (DE3) using taguchi method and efficient one-step insulin purification by on-column enzymatic cleavage.

Diabetes is a critical worldwide health problem. Numerous studies have focused on producing recombinant human insulin to address this issue. In this research, the process factors of production of recombinant His-tagged proinsulin in E. coli BL21 (DE3) strain were studied. Bacterial culture factors with significant effects on the amount of produced recombinant proinsulin were screened using a Taguchi L8 orthogonal array. Proinsulin expression was conducted under predicted optimal conditions. The folded impure His-tagged proinsulin was purified using immobilized metal ion affinity chromatography (IMAC). A novel IMAC sequence order combined with the use of non-His-tagged C-peptide cleavage enzymes followed by His- tagged enterokinase enzyme enabled simultaneous protein purification and elimination of C-peptide and His-tag in just one step. Statistical analysis revealed that the amount of produced proinsulin was significantly affected by several factors including the post-induction incubation temperature, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration, pre-induction incubation temperature, the glucose concentration, bacterial cell population at induction step, and the time of harvesting. The optimized model resulted in an empirical maximum proinsulin concentration of 254.5 ± 11.7 µg/ml. The high purity of the purified insulin (> 96% by SDS-PAGE) indicated that applied IMAC sequence order could be considered an efficient technique for on-column cleavage and insulin purification.