Yeast expression refers to a method of obtaining large quantities of a target protein by using gene cloning techniques to construct an exogenous target gene into an expression vector and then transforming it into yeast cells for stable expression. By reviewing relevant literature, this paper summarizes common issues encountered during yeast expression experiments, analyzes their possible causes, and proposes solutions.

Problem 1: When expressing a protein in Pichia pastoris, a single band resembling a trimer is observed in SDS-PAGE after small-scale expression. However, during large-scale expression, a large peak is visible on the FPLC column, yet no band is detected in the electrophoresis?

Answer: Possible reasons for observing a large peak on the FPLC column but no band in the electrophoresis include:

(1) The sample did not contain the target protein; this could be because the protein was not expressed, was not eluted during affinity chromatography, or eluted through the column;

(2) The target protein bound to the FPLC column and was not eluted;

(3) The target protein eluted through the FPLC column, and the large peak observed on the FPLC may be a salt peak;

(4) The electrophoresis was not performed properly, and the target protein was not detected.

Question 2: I used G418 to screen for high-copy cell lines following transformation. I performed two rounds of screening using four concentration gradients (0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL), but no cells grew in any of the plates, nor did the empty vector control. The vector used was pPIC9K. After electroporation and plating onto MD solid medium, I found the transformation efficiency to be low, with only about a dozen colonies growing per plate. Is this due to low transformation efficiency?

Answer: You can address this by following these steps:

(1) Pick a single yeast colony and inoculate it into a 50-ml conical flask containing 5 ml of YPD medium. Incubate overnight at 30°C and 250–300 r/min; Transfer 100–500 μl of the culture to a 2-L conical shaking flask containing 500 ml of fresh medium, and incubate overnight at 28–30°C and 250–300 r/min until the OD₆₀₀ reaches 1.3–1.5;

(2) Immediately after electroporation, add 1 ml of ice-cold 1 M sorbitol solution and mix the cells thoroughly;

(3) Recommended settings: voltage 1.5 kV; capacitance 25 μF; resistance 200 Ω; pulse duration 4–10 msec.

Question 3: I have transformed a gene into Pichia pastoris. The protein is expressed, but no positive characteristic band is detected by PCR no matter what I try. How can I improve this?

Answer: When using yeast DNA as a template, do not follow the template amount specified in the operating manual for PCR; instead, reduce the template amount—generally, a few ng is sufficient. For example, pick a single colony into 3 mL of suitable culture medium, shake the culture overnight, extract yeast DNA using a kit, and use 1 μL of the extracted product as a template for PCR verification.

Reference Procedure:

(1) Use a pipette tip or toothpick to transfer a single colony directly into 10 μL of sterile deionized water;

(2) Add 5 μL of 5 U/μL lysozyme (SIGMA);

(3) Incubate at 37°C for 30 minutes;

(4) Freeze at -70°C for 15 minutes;

(5) Boil for 5 minutes;

(6) Centrifuge at 12,000 rpm for 5 minutes;

(7) Transfer 5 μL of the supernatant to a 50 μL PCR reaction mixture.

Question 4: When expressing an antimicrobial peptide of approximately 3 kDa, the target protein is not detected by electrophoresis. What factors should be considered when expressing this peptide?

Answer: First, determine whether the protein is being secreted by checking if it is present in the bacterial cells. If not:

(1) Construct multiple secretory expression vectors to examine how various expression vector factors—such as integration mode, number of copies integrated, 5' untranslated region, and methanol utilization phenotype—affect expression levels;

(2) Generally, fermentation tanks yield higher expression levels (10–20 times) than shake flask cultures;

(3) Select yeast-preferred codons;

(4) Select protease-deficient host strains, such as SMD1168.

Question 5: When expressing proteins in Pichia pastoris (non-secretory expression), I often encounter situations where the expression level is quite high, but after cell lysis, the target protein is found in the pellet and cannot be further purified. Why is this?

Answer: When expression levels are high, the expressed protein is prone to misfolding, resulting in insoluble products. Therefore, to increase the yield of soluble protein, it is best to avoid overexpressing the target gene. Measures such as lowering the temperature can be taken.

Key Points for Competent Cell Preparation:

    The quality of glycerol and water is critical; ultrapure water is preferred. For optimal results, ensure no detergent residue remains in the wash bottles. First, fill the bottle with ultrapure water and pressurize it, then discard the water, and finally prepare a 10% glycerol solution. It is best to harvest the cells during the semi-log phase; generally, harvest at an OD of 0.5—harvesting at higher OD values yields poorer results. Directly resuspend the -80°C bacterial culture and shake it; transfer the culture the next day. Resuspending from plate colonies yields poorer results, whereas shaking the culture at 33–34°C produces more ideal results.

    When washing with an equal volume, half the volume, or one-quarter volume of ice-cold 10% glycerol, ensure the temperature is low. Gently shake during resuspension, and pour out the wash buffer completely—even if this results in some bacterial loss. Finally, approximately 500 mL of culture can yield 3–4 mL of competent cells. Freeze in liquid nitrogen for 10 minutes before transferring to a -80°C freezer; alternatively, they can be used for transformation without freezing.

    Competent cells should be tested for efficiency using a standard concentration of plasmid for electroporation; the efficiency is generally greater than 10⁹/mg. For the procedure, add 1 ng/μL of plasmid to 1 μL of competent cells in 40 μL of solution in a 0.1-cup tube. After electroporation, add 1 mL of SOC medium as quickly as possible for resuscitation, shake at 100–150 rpm for 1 hour, and plate a 1/1000 dilution; more than 1,000 colonies should grow. Only then is the sample suitable for library construction.

    Use as little plasmid or conjugate as possible during electroporation to minimize ions. In fact, if the ion concentration is too high, the current passes directly through the solution without generating an electric field, preventing the plasmid from entering the cells.

Summary:

(1) All equipment and materials must be clean to ensure the absence of ions;

(2) Maintain a constant temperature of 0°C from the moment the sample comes into contact with glycerol;

(3) Resuscitation must be performed quickly after electroporation.

    KMD Bioscience has been dedicated to recombinant protein expression and purification services for many years. We can provide you with comprehensive recombinant protein expression and purification services using yeast, including the expression vectors pPICZaA, pGAPZaA, and pPIC9K, as well as strains such as X33, GS115, and Saccharomyces cerevisiae.