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.