2.9 Recombinant protein expression and purification
To analyze the function of PpINH1 protein in vitro , thePpINH1 CDS was cloned into pET-32a at the KpnI-HindIII sites, and expressed in E. coli strain BL21 (DE3) (Beijing ComWin Biotech, China) to generate a His-fusion protein. Cells were cultured in LB medium containing 100 μg mL-1 ampicillin at 15 °C for 16 h, with shaking at 200 rpm. Expression of the recombinant protein was induced with 1.0 mM IPTG when cells reached a density 0.6 to 0.8 (OD600). The cell pellet from a 300 μL culture was lysed by sonication for 10 min in 200 μL lysis buffer (50 mM Tris, 150 mM NaCl, 5% glycerol pH 8.0). The lysate was then centrifuged at 15,000 rpm for 10 minutes, the supernatant was collected, and the PpINH1protein was purified using a Ni column.
For preparation of recombinant PpVIN2, the PpVIN2 CDS was cloned directly into a pPICZαA vector at the XhoI-NotI sites. Pichia pastoris strain X-33 was then transformed with 10 μg of the linearized construct. For small-scale evaluation of protein expression, four transformant colonies were used to inoculate buffered glycerol-complex medium (BMGY). Cells were cultured to an OD600 of 3.0, pelleted by centrifugation, and re-suspended in BMGY at 28 °C (OD600 was adjusted to 1.0). Pure methanol was added to a final concentration of 1% volume every 24 hours for 4 days. After low-speed centrifugation, the supernatant was collected and analyzed by SDS-PAGE and western blot. Protein was purified from the culture supernatants using a Ni column.
The concentrations of the purified proteins were determined using a Micro-Bradford protein assay with bull serum albumin (Thermo Fisher, USA) as a standard. For western blot analysis, purified proteins were fractionated on a 12% SDS-PAGE gel and electrotransferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). Mouse-anti-His mAb (GenScript, Nanjing, China) was used as the primary antibody. Proteins were also displayed on a Coomassie Blue-stained 12 % SDS-PAGE gel, and then protein purity was estimated by densitometric analysis.