Common Problems and Solutions in Recombinant Antibody Yeast Expression Experiments-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

Common Problems and Solutions in Recombinant Antibody Yeast Expression Experiments

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

A1. The structure of recombinant antibodies is complex, and most of them require post-translational modification, so they are suitable for expression in eukaryotic expression systems, but the cost of large-scale antibody preparation is high. The yeast expression system can perform relatively complete eukaryotic expression and is relatively simple to operate. Expression using Pichia pastoris will not accumulate ethanol, and can achieve the same degree of glycosylation as mammalian cells.

Q2. What should be paid attention to 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. Since yeast's preference for codons may affect expression efficiency, it is necessary to check whether there are rare codons in the gene. In the yeast expression system, the choice of signal peptide determines whether the recombinant antibody can be secreted correctly, so it is necessary to analyze whether a signal peptide is needed in the sequence.

Q3. What are the commonly used expression vectors for recombinant antibody yeast expression? What should be paid attention to?

A3. Commonly used yeast expression vectors include pPIC9K, pPICZaA, and pGAPZaA, among which pPIC9K is suitable for high-copy integration and stable expression. Promoters can be inserted into the expression vector to ensure efficient expression of recombinant antibodies, and Igκ signal peptides or IgH signal peptides can be used to help recombinant antibodies to be correctly localized and secreted in yeast cells. Adding His tags, FLAG tags, etc. is conducive to affinity purification of antibodies. When using a yeast expression system, the plasmid should be linearized and integrated into the genome so that the exogenous gene can exist stably.

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

A4. Common yeast host bacteria include GS115, KM71H, etc. Common transformation methods include electroporation and chemical transformation. When inducing, the induction conditions can be optimized, and the methanol concentration, temperature, pH value, expression time, etc. can be adjusted according to experimental requirements.

Q5. What problems will low transformation efficiency cause? How to deal with it?

A5. Low yeast transformation efficiency will result in the inability to obtain enough positive transformants. Generally, it is necessary to optimize the transformation conditions, such as increasing the electroporation transformation voltage, adjusting the capacitance and resistance values, ensuring that the transformation process is carried out at low temperature, and using high concentrations of plasmids and competent cells.

Q6. What problems will be encountered during the purification process? How to deal with it?

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

Q7. What problems will lead to low expression of recombinant antibodies? How to deal with it?

A7. When expressing recombinant antibodies, the selected vector and cells do not match, which may lead to low protein expression. When selecting a vector, the effects of factors such as the integration mode of the vector, the number of integration copies, and the 5' untranslated region on the expression should be considered. If the codons used are not high-frequency codons in yeast, it may affect the translation efficiency, resulting in low expression. The gene sequence can be codon optimized to improve the translation efficiency. If the yeast culture conditions such as temperature, rotation speed, culture medium, etc. are not suitable, the yeast expression may also decrease. Therefore, it is necessary to optimize the culture conditions according to the yeast species and expression requirements to ensure that the yeast is in the best growth state. In addition, there are a variety of proteases in yeast cells, which may cause the target protein to be degraded during the expression process. The degradation of the target protein can be avoided by selecting protease-deficient yeast strains or adding protease inhibitors to the culture medium.

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

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