Outline a complete strategy to produce recombinant human growth hormone (hGH) in *Escherichia coli*. Include (i) choice of vector, (ii) promoter and regulatory elements, (iii) insertion of the hGH coding sequence, (iv) selection marker, (v) steps to confirm expression, and (vi) method to purify the protein.
Answer
Use a high‑copy plasmid such as pET‑21a with the T7 promoter; clone the hGH coding sequence downstream of the promoter using compatible restriction sites, add a His‑tag for purification, select transformants on ampicillin, confirm expression by SDS‑PAGE and Western blot, and purify the His‑tagged hGH by Ni‑NTA affinity chromatography.
Step-by-step solution
- Choose a bacterial expression vector (e.g., pET‑21a) that has a strong T7 promoter and a high copy number for robust transcription.
- The T7 promoter is tightly regulated by the lac operator; IPTG can be used to induce expression when required.
- Amplify the hGH coding sequence with primers containing restriction sites compatible with the vector’s multiple cloning site, then ligate the fragment into the vector using DNA ligase.
- Incorporate an N‑terminal or C‑terminal 6×His tag in the cloning design to facilitate downstream purification.
- The vector carries an ampicillin‑resistance gene; plate transformed *E. coli* cells on LB agar containing ampicillin to select colonies that have taken up the plasmid.
- Pick positive colonies, grow a culture, induce with IPTG, and harvest cells; lyse the cells and run an SDS‑PAGE to check for a band of the expected molecular weight (~22 kDa).
- Confirm the identity of the band by Western blot using an anti‑hGH antibody.
- Purify the His‑tagged hGH from the soluble fraction using Ni‑NTA affinity chromatography, followed by dialysis into a physiological buffer.
Common mistake: Neglecting the need for a strong inducible promoter or a purification tag
Concept tested: Recombinant protein production in bacteria
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