Recombinant insulin processing — In early E. coli expression strategies, the A and B chains of insulin were expressed as fusion proteins with β-galactosidase. Which reagent was classically used to cleave the fusion at methionine residues to release the insulin chains?

Introduction / Context:Early recombinant insulin production in bacteria relied on expressing the A and B chains as part of large, stable fusion proteins (often with β-galactosidase) to enhance expression and protect peptides from degradation. A specific chemical cleavage step was then used to liberate the insulin chains cleanly for refolding and disulfide pairing.

Given Data / Assumptions:

• CNBr cleaves specifically at the carboxyl side of methionine residues in strong acid.
• Insulin A and B chains were engineered with methionine junctions to β-gal fusions.
• After cleavage, chains are purified and correctly reoxidized to form native insulin.

Concept / Approach:Cyanogen bromide is a classical reagent for site-specific chemical cleavage at methionine. By designing the fusion protein with a methionine between β-galactosidase and the insulin peptide, CNBr treatment releases the desired chain without proteolysis-induced heterogeneity associated with less specific enzymes.

Step-by-Step Solution:

Verification / Alternative check:Classical process descriptions for early recombinant insulin consistently mention CNBr cleavage of β-gal fusions, followed by oxidative folding steps to assemble biologically active insulin.

Why Other Options Are Wrong:

• Hydrogen chloride/hydroxylamine: not the standard site-specific approach for Met cleavage.
• General peptidases/proteases: lack the desired specificity and can over-digest the product.

Common Pitfalls:Assuming proteases were preferred; chemical specificity at methionine made CNBr the historical choice in these constructs.

Final Answer:Cyanogen bromide (CNBr).