pPIC-xyn11B

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	The recombinant plasmids (pPIC-xyn10A, pPIC-xyn11A, pPIC-xyn10B, and pPIC-xyn11B) were transformed into P. pastoris X-33 or GS115 through electroporation (Gene Pulser Xcell™ Electroporation System, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The transformants were plated onto YPDS plates (1% yeast extract, 2% tryptone, 2% dextrose, 1 M sorbitol, 100 μg/mL Zeocin, and 2% agar) and incubated at 30 °C for 2–3 days until colonies appeared. The P. pastoris transformants were isolated on YPDS plates containing increasing concentrations of Zeocin ranging from 1000 to 2000 μg/mL to select multicopy vector strain transformants.

Protocol tips

The recombinant plasmids (pPIC-xyn10A, pPIC-xyn11A, pPIC-xyn10B, and pPIC-xyn11B) were transformed into P. pastoris X-33 or GS115 through electroporation (Gene Pulser Xcell™ Electroporation System, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The transformants were plated onto YPDS plates (1% yeast extract, 2% tryptone, 2% dextrose, 1 M sorbitol, 100 μg/mL Zeocin, and 2% agar) and incubated at 30 °C for 2–3 days until colonies appeared. The P. pastoris transformants were isolated on YPDS plates containing increasing concentrations of Zeocin ranging from 1000 to 2000 μg/mL to select multicopy vector strain transformants.

Publication protocol

The recombinant plasmids (pPIC-xyn10A, pPIC-xyn11A, pPIC-xyn10B, and pPIC-xyn11B) were transformed into P. pastoris X-33 or GS115 through electroporation (Gene Pulser Xcell™ Electroporation System, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The transformants were plated onto YPDS plates (1% yeast extract, 2% tryptone, 2% dextrose, 1 M sorbitol, 100 μg/mL Zeocin, and 2% agar) and incubated at 30 °C for 2–3 days until colonies appeared. The P. pastoris transformants were isolated on YPDS plates containing increasing concentrations of Zeocin ranging from 1000 to 2000 μg/mL to select multicopy vector strain transformants. Twenty colonies from the 2000 μg/mL Zeocin YPDS plates were inoculated into 10 mL of YPM (1% yeast extract and 2% tryptone) medium containing 1% methanol in a 50-mL flask and cultured at 28 °C on a rotary incubator at 250 rpm to induce enzyme expression. The enzymatic activity of each supernatant was analyzed to determine the transformant with the best secretion yield for each enzyme. The transformants with the highest xylanase activity in a culture supernatant following 3–4 days of incubation were used for further fermentation in 1-L flasks following the method of Bai et al.44. The culture supernatants were recovered after 3–4 days of methanol induction through centrifugation (10 min, 5,000 × g), and the suspensions were concentrated using a 10-kDa molecular weight cut-off (MWCO) ultrafiltration membrane (Sartorius, Göttingen, Germany). The expressed 6 × His-tagged proteins were further purified using Ni-NTA Sepharose (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

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