Outline a complete strategy to express recombinant human growth hormone (hGH) in Chinese Hamster Ovary (CHO) cells using a mammalian expression system. 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) purification method for the secreted protein.

Step-by-step solution

1. i) Choose a high‑copy mammalian vector such as pcDNA3.1(+) that is known to replicate in CHO cells.
2. ii) Use the strong cytomegalovirus (CMV) immediate‑early promoter to drive high transcription; include a Kozak consensus sequence ($GCCACC$) upstream of the start codon for efficient translation and a polyadenylation signal (SV40 polyA) downstream for mRNA stability.
3. iii) Insert the hGH coding sequence (≈1,000 bp) downstream of the promoter, preceded by a native or engineered signal peptide (e.g., the IgG $\kappa$ leader) to direct secretion into the culture medium; ligate using compatible restriction sites and confirm orientation by colony PCR and sequencing.
4. iv) Incorporate the neomycin resistance gene (neo) as a selectable marker; after transfection, add G418 to the medium so only cells that have integrated the plasmid survive.
5. v) Confirm expression: (a) isolate total RNA from G418‑resistant clones and perform RT‑PCR to detect hGH transcripts; (b) collect culture supernatant and run SDS‑PAGE followed by Western blot using anti‑hGH antibodies; (c) quantify secreted hGH by ELISA to select high‑producing clones.
6. vi) Purify the secreted hGH: if a C‑terminal 6×His tag was engineered, apply the supernatant to a Ni‑NTA agarose column, wash with low‑imidazole buffer, elute with high‑imidazole buffer, and finally dialyse against physiological buffer. Alternatively, use immunoaffinity chromatography with anti‑hGH antibodies.

Concept tested: Recombinant protein production in mammalian cells

Published 1 October 2026 · How this solution was prepared and checked