Prompt Description

The overall process for extracting and purifying recombinant human insulin from E. coli inclusion bodies is as follows: First, inclusion bodies containing proinsulin fusion protein are obtained through fermentation and induced expression. Subsequently, cells are lysed, and the inclusion bodies are recovered, followed by washing to remove impurities. Next, the inclusion bodies are dissolved using a high concentration of denaturant (such as urea) and subjected to oxidative sulfitolysis to maintain the proinsulin in an unfolded state and prevent incorrect disulfide bond formation. Then, the N-terminal fusion peptide is cleaved using cyanogen bromide (CNBr). This is followed by buffer exchange to remove reaction reagents. Proinsulin is then purified by chromatographic methods (such as ion exchange, reversed-phase chromatography, etc.) and/or methods like pH precipitation and zinc crystallization. After purification, refolding is performed to restore its native conformation. Subsequently, the C-peptide is removed by enzymatic digestion using trypsin and carboxypeptidase B, generating the biologically active two-chain insulin structure. Finally, further chromatographic purification and formulation steps are performed to obtain high-purity recombinant human insulin or analog products.