I. Determination of Mut+ and Muts Phenotypes

After the transformants have grown on the plates for a period of time, screen for Mut+ and Muts phenotypes. Pick single colonies and streak or spot them on MM and MD media (first spot on MM plates, then on MD plates, using a new toothpick for each clone). Incubate at 30°C for 2 days. Observe the growth: colonies that grow on MD medium but not (or very poorly) on MM medium are identified as the Muts phenotype, while the rest are classified as Mut+ phenotype.

II. Induction Expression Experiment of Mut+ Phenotype Recombinant Yeast

1.Pick a single colony and inoculate it into a 250 ml flask containing 25 ml of MGY, BMG, or BMGY medium. Culture at 28-30°C with shaking at 250-300 rpm until OD600 reaches 2-6 (approximately 16-18 hours).

2.Centrifuge the culture at 1500-3000g for 5 min at room temperature to harvest the cells. Resuspend the cell pellet in MM, BMM, or BMMY to an OD600 of ~1.0.

3.Transfer the resuspended culture from step 2 into a 1 L flask, cover the opening with double-layered gauze or cheesecloth, and continue incubation at 28-30°C with shaking at 250-300 rpm.

4.Every 24 hours, add 100% methanol to the culture to a final concentration of 0.5-1.0%.

5.Collect samples (1 ml each) at specified time points (typically 0, 6, 12, 24, 36, 48, 60, 72, 84, and 96 hours) in 1.5 ml microcentrifuge tubes. Centrifuge at maximum speed for 2-3 min, then separate the supernatant and cell pellet to analyze target protein expression levels and determine the optimal harvest time.

6.For secreted expression, isolate the supernatant from the samples. For intracellular expression, isolate the cell pellet. Flash-freeze the samples in liquid nitrogen or dry ice and store at -80°C for further analysis.

7.Recombinant protein expression can be detected and verified by SDS-PAGE, Western blot, and activity assays.

III. Induced Expression of Recombinant Yeast with Mut Phenotype

1.Pick a single colony and inoculate it into a 250 mL flask containing 25 mL of MGY, BMG, or BMGY medium. Culture at 28–30 °C with shaking at 250–300 rpm until OD₆₀₀ reaches 2–6 (approximately 16–18 h).

2.Centrifuge the culture at 1,500–3,000 × *g* for 5 min at room temperature to harvest the cells. Resuspend the cell pellet in 1/5 to 1/10 of the original culture volume (approximately 10–20 mL) using MM, BMM, or BMMY medium.

3.Transfer the resuspended culture from Step 2 into a 100 mL flask, cover the opening with double-layered gauze or cheesecloth, and continue incubation at 28–30 °C with shaking at 250–300 rpm.

4.Every 24 h, add 100% methanol to the culture to a final concentration of 0.5–1.0%.

5.Collect 1 mL samples at specified time points (typically 0, 24, 48, 72, 96, and 120 h) in 1.5 mL microcentrifuge tubes. Centrifuge at maximum speed for 2–3 min, then separate the supernatant and cell pellet to analyze target protein expression levels and determine the optimal harvest time.

6.For secretory expression, isolate the supernatant from the samples. For intracellular expression, isolate the cell pellet. Flash-freeze the samples in liquid nitrogen or dry ice and store at −80 °C for further analysis.

7.Recombinant protein expression can be detected and verified by SDS-PAGE, Western blot, and activity assays.

IV. KMD Bioscience Offers Recombinant Protein Yeast Expression Services

KMD Bioscience has years of dedication to recombinant protein expression and purification services. We provide comprehensive yeast expression and purification solutions, utilizing expression vectors such as pPICZaA, pGAPZaA, and pPIC9K, along with host strains including X33, GS115, and Saccharomyces cerevisiae.