Pichia pastoris gs115 cells

Wild-type (WT) TrCel12A (Egl3) with signal peptide excluded and the 6 ×His-tag designated at the C-terminal was cloned and inserted into pPIC9k (Invitrogen, Carlsbad, CA, USA), while mutants were constructed by site-directed mutagenesis according to the PCR-based method54 (link). Competent yeast (Pichia pastoris) GS115 cells (Invitrogen) were then transformed with the recombinant plasmids after they were confirmed by DNA sequencing (Biosune, Shanghai, China). The exogenous proteins were induced and purified with the protocols described in the Original Pichia Expression Kit (Invitrogen) and the QIAexpress Kit (Qiagen, Hilden, Germany). All proteins expressed were buffer exchanged in 20 mM acetate buffer (pH 5.5) and the concentration was determined by the Bradford method55 (link). Manipulations to another member of the GH12 family, AnEglA, from Aspergillus niger CBS120.49 (PDB 1KS4) were the same to TrCel12A and the WT AnEglA and its mutants were buffer exchanged in the same acetate buffer with pH 3.8. All chemicals, reagents and enzymes used were of analytical grade from Sigma (St. Louis, MO, USA) or Sangon (Shanghai, China).

The plasmid containing the OfChtI-CAD gene was transformed into Pichia pastoris GS115 cells (Invitrogen, Carlsbad, California, USA) for the overexpression of recombinant OfChtI-CAD. The cells were first grown in buffered glycerol complex medium (BMGY; 1% yeast extract, 2% peptone, 1% glycerol, 1.34% yeast nitrogen, 0.2% biotin, 100 mM potassium phosphate pH 6.0) at 303 K to an optical density (OD) of 2.0 at 600 nm. After reaching this OD, the cells were collected and resuspended in buffered methanol complex medium (BMMY; 1% yeast extract, 2% peptone, 1% methanol, 1.34% yeast nitrogen, 0.2% biotin, 100 mM potassium phosphate pH 6.0). Methanol [1%(v/v)] was added at 24 h intervals as an inducer. After 72 h of fermentation, the culture supernatant was harvested and subjected to ammonium sulfate precipitation (75% saturation) at 277 K. The precipitate was dissolved in buffer A (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole pH 7.4) and the sample was then loaded onto a HisTrap HP affinity column (5 ml; GE Healthcare, USA) pre-­equilibrated with buffer A. After washing the column, the target protein was eluted with buffer B (20 mM sodium phosphate, 0.5 M NaCl, 200 mM imidazole pH 7.4). The purity of the eluted protein was analyzed by SDS–PAGE and found to be >95%. The mutants were expressed and purified using the same procedure as used for wild-type OfChtI-CAD.

Site-directed mutagenesis kit was purchased from Trans Gen (Beijing, China). Restriction endonuclease (EcoR I, Not I), DNA marker, and protein marker were purchased from TaKaRa (Otsu, Japan). Pichia pastoris GS115 cells were purchased from Invitrogen (Shanghai, China). Mutant primers were synthesized by Shuoqing (Kunming, China). The Thermomyces lanuginosus lipase gene (TLL) cloned in the pPIC9K vector was deposited in our laboratory. All other chemicals were commercially available and of analytical grade.