pPIC9-His-FIP-Ganoderma

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	Preparation and transformation of yeast competent cells were performed according to the kit instruction (Catalog number: T2001). Phenotype of yeast transformants was determined by screening on MD and MM media. Positive transformants were verified by colony PCR and cultured in YPD at 30 °C, 200 rpm until OD600 ≥ 2.0.

Protocol tips
Preparation and transformation of yeast competent cells were performed according to the kit instruction (Catalog number: T2001). Phenotype of yeast transformants was determined by screening on MD and MM media. Positive transformants were verified by colony PCR and cultured in YPD at 30 °C, 200 rpm until OD600 ≥ 2.0.

Publication protocol

Preparation and transformation of yeast competent cells were performed according to the kit instruction (Catalog number: T2001). Phenotype of yeast transformants was determined by screening on MD and MM media. Positive transformants were verified by colony PCR and cultured in YPD at 30 °C, 200 rpm until OD600 ≥ 2.0. Then the yeast cells were harvested by centrifugation at 6000 rpm for 5 min and then resuspended in BMMY 200 mL 500 mL− 1 in one flask) to induce protein expression. Methanol was added every 24 h to maintain a final concentration of 1% (v/v). At 96 h, 1 mL of the expression cultures were transferred for later use. The wild-type yeast was used as a negative control and cultured in a same way. Purification of recombinant Ganoderma FIPs was carried out using HisTrap™ FF prepack columns according to the manufacturer’s protocol (Catalog number: 17–5319-01). The protein masses of recombinant proteins were determined through SDS–PAGE and quantitative analyses with an image-analyzing system (QUANTITY ONE, Bio-Rad, USA) [28].

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