Expression and Purification of Recombinant Proteins in Escherichia coli

For ectopic overexpression in E. coli, the open reading frames of PbTPSs were amplified and cloned into the expression vector pET-28b (Novagen, Madison, WI, United States), which has a His-tag on both the N- and C-termini of the inserted fragment. The construct was introduced into E. coli C41 (DE3) pLysS strain (Invitrogen, Carlsbad, CA, United States). Sequence analysis and protein structure modeling confirmed that no mutant occurred near the protein active site on DNA amplification. A single colony of the constructs was incubated in 5 ml LB medium with 50 μl/ml kanamycin overnight at 37°C. Then, the culture was transferred to 750 ml LB medium with 50 μl/ml kanamycin, incubated at 37°C to OD₆₀₀ = 0.4–0.6, and induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown under 16°C for 22 h. Cells were pelleted by centrifugation and suspended in extraction buffer (0.3 M NaCl, 50 mM HEPES, pH 7.4), then disrupted by sonication (SONICS, VC750, Newtown, CT, United States). Cell extracts were filtered through a 0.45-μm filter. Recombinant proteins were purified by using HiTrap TALON crude (GE Healthcare, Abingdon, United Kingdom) with different concentrations of imidazole solution (0.3 M NaCl, 50 mM HEPES, 5 mM/150 mM imidazole, pH 7.4). Protein expression was verified on Coomassie Blue-stained SDS–PAGE gel and Western blot analysis with 1/10,000 diluted mouse anti-histidine monoclonal antibody (Roche Diagnostics, Penzberg, Germany).