Recombinant insulin design — Which approach correctly describes a classic method for synthesizing human insulin using recombinant DNA technology?
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AUsing chemically synthesized DNA sequences for the A and B chains separately (expressed as fusion proteins and then recombined)
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BIsolating genomic DNA directly from the islets of Langerhans and expressing it intact
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CUsing cDNA for insulin but expressing it without any processing or proinsulin strategy
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DUsing a single chemically synthesized DNA sequence for the complete insulin protein with no subsequent processing
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EExtracting mature insulin directly from human plasma donors
Answer
Correct Answer: Using chemically synthesized DNA sequences for the A and B chains separately (expressed as fusion proteins and then recombined)
Explanation
Introduction / Context:Recombinant human insulin was produced using early bacterial expression strategies that overcame challenges of peptide stability and processing. Understanding the chain-by-chain method explains how bioidentical insulin was obtained without relying on animal pancreases.
Given Data / Assumptions:
- Insulin comprises A and B polypeptide chains linked by disulfide bonds.
- Bacteria do not process preproinsulin like human beta cells.
- Fusion expression and chemical cleavage enabled high yields and purity.
Concept / Approach:One classic strategy used two synthetic genes (for A and B chains). Each chain was expressed in E. coli as a β-galactosidase fusion, cleaved with cyanogen bromide, purified, and then oxidatively refolded together to form native disulfide bonds. Alternative approved methods later used proinsulin cDNA with enzymatic processing, but the chain-by-chain method is historically significant and matches the option given.
Step-by-Step Solution:
Chemically synthesize DNA for A and B chains.Express each as a stable fusion in E. coli.Cleave fusions (e.g., CNBr at Met), purify chains.Reconstitute A+B with correct disulfide formation to yield insulin.Verification / Alternative check:Regulatory and historical accounts of Humulin describe chain-by-chain and proinsulin strategies; both yield bioidentical insulin but the option specifying separate chain synthesis matches the classic approach.
Why Other Options Are Wrong:
- Genomic DNA from islets: includes introns and regulatory sequences unsuitable for direct bacterial expression.
- cDNA without processing: proinsulin requires proper cleavage; expression systems must include processing steps.
- Single synthetic gene with no processing: still requires folding/processing; oversimplified.
- Plasma extraction: not feasible for insulin.
Common Pitfalls:Assuming bacteria can process preprohormones like human cells; engineered strategies are required to obtain mature insulin.
Final Answer:Using chemically synthesized DNA sequences for the A and B chains separately (expressed as fusion proteins and then recombined).