Outline a complete strategy to produce recombinant SARS‑CoV‑2 spike protein in the baculovirus–insect cell expression system. Include (i) choice of vector, (ii) promoter and regulatory elements, (iii) insertion of the spike coding sequence, (iv) selection marker, (v) steps to confirm expression, and (vi) method to purify the protein.
Answer
A step‑wise plan covering vector, promoter, cloning, selection, confirmation and purification as described below.
The correct answer is A step‑wise plan covering vector, promoter, cloning, selection, confirmation and purification as described below. — Validate the purified protein by ELISA and circular dichroism to ensure correct folding.
Step-by-step solution
- Choose a baculovirus transfer vector such as pFastBac™1, which contains the Tn7 transposition site for easy insertion into the bacmid.
- Use the strong polyhedrin promoter in the vector to drive high‑level expression in insect cells; include a downstream SV40 polyadenylation signal for mRNA stability.
- Amplify the spike gene (with a suitable signal peptide for secretion) by PCR, add BamHI and XhoI sites, and ligate into the multiple‑cloning site downstream of the polyhedrin promoter.
- The vector carries a kanamycin resistance gene for selection in E. coli and a lacZα reporter for blue‑white screening; the bacmid carries a chloramphenicol marker for selection in DH10Bac cells.
- Transform the recombinant plasmid into DH10Bac E. coli, select white colonies on kanamycin/X‑gal/IPTG plates, and isolate bacmid DNA containing the spike insert.
- Transfect purified recombinant bacmid into Sf9 insect cells using a lipid‑based reagent; allow virus replication and amplify the recombinant baculovirus (P1 → P2 stock).
- Infect a larger culture of Sf9 or High‑Five cells with the P2 virus; harvest the culture medium after 72 h because the spike protein is secreted.
- Confirm expression by (a) PCR on viral DNA, (b) SDS‑PAGE of the medium showing a ~180 kDa band, and (c) Western blot using anti‑spike antibodies.
- Purify the secreted spike protein using Ni‑NTA affinity chromatography (the construct includes a C‑terminal His₆ tag); perform size‑exclusion chromatography for final polishing.
- Validate the purified protein by ELISA and circular dichroism to ensure correct folding.
Common mistake: Omitting the signal peptide, which prevents secretion of the spike protein into the medium
Concept tested: Recombinant protein production in baculovirus system
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