Common Problems and Solutions in Recombinant Antibody Yeast Expression Experiments

Q1. Under what circumstances is it appropriate to use yeast protein expression systems for antibody recombinant expression?

A1. The structure of recombinant antibodies is complex and most require post-translational modifications, making them suitable for expression in eukaryotic expression systems. However, large-scale production of recombinant antibodies is costly., The yeast protein expression systems can achieve relatively complete eukaryotic expression and are relatively easy to operate. Using Pichia pastoris for expression does not accumulate ethanol and can achieve the same degree of glycosylation as mammalian cells.

Q2. What should be noted when analyzing antibody sequences?

A2. The analysis of the gene sequence of recombinant antibodies should be as detailed as possible to ensure the integrity and accuracy of the gene sequence. Due to the potential impact of yeast's preference for codons on expression efficiency, it is necessary to investigate the presence of rare codons in genes. In the yeast expression system, the selection of signal peptides determines whether recombinant antibodies can be secreted correctly, so it is necessary to analyze whether signal peptides are needed in the sequence.

Q3. What are the commonly used expression vectors for recombinant antibody yeast expression?

A3. Commonly used yeast expression vectors include pPIC9K pPICZaA、pGAPZaA， Among them, pPIC9K is suitable for high copy integration and stable expression. Promoters can be inserted into expression vectors to ensure efficient expression of recombinant antibodies, and Ig κ or IgH signal peptides can be used to assist in the correct localization and secretion of recombinant antibodies in yeast cells. Adding His tags, FLAG tags, etc. is beneficial for affinity purification of antibodies. When using yeast expression systems, plasmids should be linearized and integrated into the genome to ensure the stable existence of exogenous genes.

Q4. What are the commonly used yeast host bacteria for transformation? What should be noted for subsequent induction expression?

A4. Commonly used yeast host bacteria include GS115, KM71H, etc. The commonly used conversion methods include electric shock conversion and chemical conversion. During induction, the induction conditions can be optimized by adjusting methanol concentration, temperature, pH value, expression time, etc. according to experimental requirements.

Q5. What problems can low conversion efficiency lead to? How should we respond?

A5. Low yeast conversion efficiency can result in insufficient positive transformants being obtained. Generally, it is necessary to optimize the transformation conditions, such as increasing the electric shock transformation voltage, adjusting the capacitance and resistance values, ensuring that the transformation process is carried out at low temperatures, and using high concentrations of plasmids and competent cells.

Q6. What problems will be encountered during the purification process? How should we respond?

A6. The target protein is prone to denaturation or degradation during or after purification. During the purification process, low-temperature operation should be maintained, stabilizers such as glycerol and sucrose should be added, and appropriate buffer solutions and pH values should be selected to protect the stability of the protein.

Q7. What issues can lead to low expression levels of recombinant antibodies? How should we respond?

A7. When selecting a vector for recombinant antibody expression that does not match the cells, it may lead to low protein expression levels. When selecting a vector, factors such as the integration method, integration copy number, and 5 'untranslated region should be considered to affect expression levels. If the codon used is not a high-frequency codon in yeast, it may affect translation efficiency and lead to lower expression levels. Therefore, codon optimization can be performed on gene sequences to improve translation efficiency. If the cultivation conditions for a series of yeast, such as temperature, rotation speed, and culture medium, are not suitable, it may also lead to a decrease in yeast expression. Therefore, it is necessary to optimize the cultivation conditions according to the yeast type and expression requirements to ensure that the yeast is in the best growth state. In addition, there are various proteases present in yeast cells that may lead to the degradation of the target protein during expression. This can be avoided by selecting protease deficient yeast strains or adding protease inhibitors to the culture medium.

KMD Bioscience has rich experience in recombinant antibody yeast expression services and recombinant antibody production, and can provide customers with protein fermentation services of various scales. With our comprehensive protein purification platform, we can provide customers with high-quality antibody discovery services in a short period of time. KMD Bioscience has a complete and mature yeast expression system, providing one-stop services from gene synthesis to yeast expression and antibody purification. We have various expression vectors such as pPICZaA, pGAPZaA, pPIC9K, as well as strains such as X33, GS115, and brewing yeast. With large-scale fermentation, we can provide industrial grade services. At the same time, we are equipped with multiple protein expression and purification methods to meet customers' different fragment antibody expression and recombinant antibody expression needs, and customize complete antibody discovery solutions for customers.

Reference

[1] Das PK, Sahoo A, Veeranki VD. Recombinant monoclonal antibody production in yeasts: Challenges and considerations. Int J Biol Macromol. 2024;266(Pt 2):131379.

[2] Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15(6):553-7.

[3] Liu CP, Tsai TI, Cheng T, et al. Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation. Proc Natl Acad Sci U S A. 2018;115(4):720-725.