pET-21b-VP24

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	All recombinant plasmids were transformed into Escherichia coli BL21 (DE3) cells. Overexpression was performed by the induction of mid-log-phase E. coli BL21 (DE3) cells with 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 12–16 h at 16°C.	The overexpressed VP24-His6 was purified using Ni–NTA affinity chromatography (GE Healthcare) followed by gel filtration using Superdex 200 HR 16/60 (GE Healthcare) equilibrated in a buffer consisting of 25 mM Tris–HCl pH 7.0, 200 mM NaCl, 5% glycerol.

Protocol tips
All recombinant plasmids were transformed into Escherichia coli BL21 (DE3) cells. Overexpression was performed by the induction of mid-log-phase E. coli BL21 (DE3) cells with 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 12–16 h at 16°C.
Downstream tips
The overexpressed VP24-His6 was purified using Ni–NTA affinity chromatography (GE Healthcare) followed by gel filtration using Superdex 200 HR 16/60 (GE Healthcare) equilibrated in a buffer consisting of 25 mM Tris–HCl pH 7.0, 200 mM NaCl, 5% glycerol.

Publication protocol

The gene coding for the transmembrane region-truncated construct of VP24 (residues 27–208; Table 1▸) was amplified from the WSSV genome by PCR using specific primers: forward primer GCCTCATATGAACATAGAACTTAAC and reverse primer GCCCTCGAGTTTTTCCCCAACC. The PCR products were digested with NdeI and XhoI and further cloned into pET-21b, resulting in a C-terminal hexahistidine tag for purification. The mutants (L69M, L88M, L121M, I133M and L69M/L121M) were generated by site-directed mutagenesis using the pET-21b-VP24 vector as the template and were confirmed by sequencing. All recombinant plasmids were transformed into Escherichia coli BL21 (DE3) cells. Overexpression was performed by the induction of mid-log-phase E. coli BL21 (DE3) cells with 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 12–16 h at 16°C. The overexpressed VP24-His6 was purified using Ni–NTA affinity chromatography (GE Healthcare) followed by gel filtration using Superdex 200 HR 16/60 (GE Healthcare) equilibrated in a buffer consisting of 25 mM Tris–HCl pH 7.0, 200 mM NaCl, 5% glycerol. Purified protein fractions were made 0.5 M in the nondetergent sulfobetaine 3-(1-pyridino)-1-propanesulfonate (NDSB-201; Sigma) and then concentrated using centrifugal concentrators to a final concentration of 2.5–3.5 mg ml−1; the concentration was measured using a Bradford Protein Assay kit (Bio-Rad). Selenomethinonine (SeMet)-labelled proteins were produced by inhibiting endogenous methionine biosynthesis in M9 minimal medium supplemented with specific amino acids as well as SeMet (Qoronfleh et al., 1995 ▸; Hendrickson et al., 1990 ▸). The purification and concentration steps were the same as for the corresponding native protein. The purification-step results, shown by SDS–PAGE, are shown in Figs. 1▸ and 2▸. Macromolecule-production information is summarized in Table 1▸.

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