Yeast protein extraction reagent

Yeast two-hybrid (Y2H) assays were performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Each pair of indicated genes were cloned into pGBKT7-DEST for fusing with GAL4 DNA binding domain (BD) or pGADT7-DEST for fusing with GAL4 activation domain (AD). Yeast competent cells (Y2H Gold) was co-transformed with bait and prey constructs, followed by 10-fold serial dilution and plating onto synthetic defined (SD) yeast leucine and tryptophan dropout medium (SD/-Leu/-Trp) or leucine, tryptophan, histidine and adenine dropout medium (SD/-Leu/-Trp/-His/-Ade). The transformants were allowed by 4- to 6-day growth on the dropout mediums at 28°C. For immunoblot detection of protein expression in yeast, the co-transformed yeast cells were propagated in liquid SD/-Leu/-Trp medium, and the cultures were harvested at OD₆₀₀ of 0.5. The total proteins were extracted by using Yeast Protein Extraction Reagent (Takara), followed by immunoblot detection with anti-HA monoclonal or anti-Myc polyclonal antibody (Abcam). Membrane yeast two hybrid (MYTH) assays were exactly performed according to the user manual of DUALmembrane starter kits (Dualsystems Biotech). Yeast competent cells (NMY51) were co-transformed with each pair of the indicated constructs, and plated onto SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade mediums.

Cells were grown in the fermentation medium with xylose as carbon source and harvested in the exponential growth phase. Cells were washed twice with sterile water, and proteins were extracted using yeast protein extraction reagent (Takara, Beijing, China) according to the manufacturer’s instructions. Protein concentrations were measured using Bradford protein assay kit (Takara, Beijing, China). XI and XDH activities were measured according to the method described by Dmytruk et al. [15 (link)]. One unit of XI activity is defined as the amount of enzyme that produces 1 μmol of xylulose per min. One unit of XDH activity is defined as micromoles of NADH (nicotinamide adenine dinucleotide plus hydrogen) reduced or oxidized produced by 1 mg of protein per min. XR activity was measured according to the method described by Bengtsson et al. [16 (link)]. One unit of XD activity is defined as micromoles of NADH reduced or oxidized produced by 1 mg of protein per min.

The sequence of pglA, excluding signal peptide sequence (1–81 bp), was obtained through direct gene synthesis. The resulting fragment was subsequently inserted into the pGBKT7 vector. The recombinant vector, pGBKT7-pglA, was then introduced into Saccharomyces cerevisiae strain Y2HGold using Yeastmaker^TM Yeast Transformation System 2 (Clontech, CA, USA). To evaluate the potential toxicity and autoactivation of the pglA bait, the transformed cells were plated onto three distinct media of SD/-Trp, SD/-Trp/X, and SD/-Trp/X/A, respectively. Following plating, the cells were cultured at 30 °C for 3–5 days. The strain of Y2HGold containing the pGBKT7-BD vector was spread on an SD-Trp medium, as a positive control.
To determine the expression of pglA bait, the transformed cells were cultured in an SD/-Trp broth medium at 30 °C under 200 rpm for 4–8 h, until the OD₆₀₀ value reached the range of 0.4–0.6. As positive control, the Y2HGold strain carrying the pGBKT7-53 vector and the Y2HGold strain carrying the pGBKT7-BD vector were also cultured in an SD/-Trp broth medium. For negative control, the wild type Y2HGold strain was cultured in a YPDA broth medium. The various yeast cells were harvested via centrifugalization, and the total protein was extracted using the Yeast Protein Extraction Reagent (Takara, Dalian, China).