Isolation of Bioactive Insulin via C-Chain Cleavage and Inclusion Body-Based Purification from E. coli BL21-(DE3) v1

This protocol describes the post-expression processing of recombinant human insulin in E. coli BL21 (DE3), focusing on the cleavage of the C-chain to obtain functional insulin composed of A and B chains. Following optimal expression using 0.5 mM IPTG in Terrific Broth, the cells were lysed via high-pressure homogenization. Inclusion bodies containing proinsulin were isolated, washed with Triton X-100 buffer, and prepared for downstream solubilization and refolding. The C-chain was cleaved during the refolding process, yielding biologically active insulin. Final purification and lyophilisation produced stable, functional insulin suitable for further biochemical and structural characterisation.