Ni ion affinity chromatography

A synthetic gene encoding the mature sequence (with the sole addition of the initial methionine) of PBP1 of B. mori (acc. no. P34174) was subcloned in the expression vector pET15b (Novagen, Darmstadt, Germany), along with standard procedures, allowing the protein to be expressed as a fusion construct bearing a polyhistidine (his-tag) at the amino terminus. Following selection of the colonies containing the insert performed by PCR analysis, positive clones were used to express the protein in Escherichia coli, following a standard protocol. After sonication, the protein was mainly present in the pellet and was solubilized in 8 M urea and 1 mM dithiothreitol (DTT). Refolding was accomplished by extensive dialysis (three times overnight with changes of buffer) against 50 mM Tris-HCl, pH 7.4. The PBP1 was purified by affinity chromatography on Ni ion affinity chromatography (GE Healthcare) along with the manufacturer’s protocol. The purity of the protein was assessed by SDS-PAGE (inset of Figure 1A).

Recombinant pET30a(+)/CSP1 and pET30a(+)/CSP2 were generated by ligating the sticky ends of the designed HoblCSP1 and HoblCSP2 constructs into the expression vector pET30a (Novagen, Germany). pET30a(+)/CSP1 and pET30a(+)/CSP2 were then transformed into E. coli BL21 (DE3) pLysS cells. The expressions of recombinant HoblCSP1 and HoblCSP2 were induced for 4 h by 0.5 mM IPTG following a 3 h pre-incubation. The cells were harvested by centrifugation and then homogenized in phosphate-buffered saline (PBS, 0.04 M, pH 7.0). After centrifugation at 12,000×g for 20 min at 4°C, the supernatants were purified by Ni ion affinity chromatography (GE-Healthcare). Recombinant HoblCSP1 and HoblCSP2 were identified by western blot analysis with antibodies designed against the His-tag (Abcam, USA). To prevent confounding effects in the subsequent experiments, the His-tag was removed by recombinant enterokinase (rEK) (Bio Basic Inc.) and NaCl was removed by dissolving the proteins in dH₂O and filtering with a 10 kDa Amicon Ultra-0.5 Device (Millipore, USA). Finally the recombinant proteins were stored at −80°C, until required.