Outline a complete strategy to produce recombinant human insulin in Escherichia coli. Include (i) choice of vector, (ii) promoter and regulatory elements, (iii) insertion of the insulin coding sequence, (iv) selection marker, (v) steps to confirm expression, and (vi) method to purify the protein.

Step-by-step solution

1. Step 1: Choose a high‑copy plasmid such as pET‑21b(+) that contains a T7 promoter and a kanamycin resistance gene for selection.
2. Step 2: Incorporate a lac operator downstream of the T7 promoter to allow IPTG‑induced control of transcription.
3. Step 3: Clone the human insulin cDNA into the multiple‑cloning site of pET‑21b using appropriate restriction enzymes (e.g., NdeI and XhoI). Verify the insert by sequencing.
4. Step 4: Transform the recombinant plasmid into an E. coli strain that expresses T7 RNA polymerase (e.g., BL21(DE3)). Select transformants on kanamycin plates.
5. Step 5: Induce protein expression by adding IPTG to mid‑log phase cultures and incubate under optimal temperature (e.g., 37 °C for 3 h). Harvest cells and lyse to extract proteins.
6. Step 6: Confirm insulin expression by running an SDS‑PAGE gel and probing with an anti‑insulin antibody in a western blot to verify correct molecular weight (~6 kDa).
7. Step 7: Purify the His‑tagged insulin by immobilized metal ion affinity chromatography (IMAC) using a Ni²⁺ column; elute with imidazole gradient.
8. Step 8: Further purify by size‑exclusion chromatography if required, and verify purity by HPLC or mass spectrometry.
9. Step 9: Verify bioactivity of purified insulin by a glucose uptake assay in cultured cells.
10. Step 10: Store purified insulin at −80 °C in aliquots to maintain activity.

Concept tested: Recombinant protein production

Published 23 September 2026 · How this solution was prepared and checked