3.6 Expression of PpINH1 and PpVIN2 recombinant proteinsin vitro
The 438 bp PpINH1 gene encodes a protein of 145 amino acids with a theoretical molecular mass of 15.06 kDa and a predicted isoelectric point of 4.34. PpINH1 is not predicted to contain a signal peptide (Figure S1a ) or a transmembrane region (Figure S2a ). The PpINH1 cDNA was cloned into pET-32a and expressed as an N-terminal His-tagged protein in E. coli BL21 (DE3). After induction conditions were optimized (data not shown), expression was induced at 15 °C for 16 h and the protein was recovered in the supernatant (Figure 5a, b ). The yield was about 17.7 mg per liter of culture.
The PpVIN2 CDS is 2061 nucleotides in length and encodes a protein of 686 amino acids, with a predicted molecular mass of 76.49 kDa and an isoelectric point of 6.45. PpVIN2 is not predicted to contain a signal peptide (Figure S1b ). The C-terminus contains a potential transmembrane region (Figure S2b ). The molecular weight of the protein after removal of the transmembrane domain is 64 kDa. The addition of the His-tag results in a 73.1 kDa fusion protein (Figure 5e, f ). To conduct biochemical and molecular studies,PpVIN2 was expressed in Pichia pastoris using the pPICZαA vector. After expression was induced with methanol, the recombinant protein was purified from the supernatant, with a yield of about 2.77 mg per liter of culture. Samples of purified PpINH1-5×His (32.4 kDa) and PpVIN2-5×His (73.1 kDa) are displayed on polyacrylamide gels inFigure 5c, d and Figure 5g, h , respectively.