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

Common Problems and Solutions for Recombinant Antibody Yeast Expression Experiments

I. What types of yeast are commonly used for recombinant antibody expression?

Pichia pastoris is the main yeast used for the production of recombinant antibodies. Other yeasts such as Saccharomyces cerevisiae, Saccharomyces polymorpha, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Kluyveromyces lactis, and Yarrowia lipolytica can also produce proteins, but are not usually preferred.

Pichia pastoris metabolizes methanol as the only carbon source. The alcohol oxidase 1 (AOX1) promoter is strictly controlled by methanol and is often used for the expression of recombinant proteins. The secretory production of exogenous proteins, including antibodies, requires an amino-terminal signal sequence that targets the yeast secretion pathway. The yeast fermentation factor α (α-factor) pre-propeptide is the most commonly used secretion signal sequence, followed by an appropriate proteolytic cleavage site that is sensitive to the Golgi endogenous protease KEX2 to effectively release the antibody during secretion.

II. What is the expression yield of different antibody fragments in the fermentation system?

Antibody stability and pharmacokinetics By binding to various receptors, the region has a modular structure. These regions of antibodies produced from different sources can be individually engineered and combined to make new antibodies with different properties. This "divide and conquer" strategy has become a common approach in antibody engineering. The functions of different antibody structures are shown in Figure 1.

The yields of different scFvs range from 70 mg/L to 250 mg/L. Under optimized conditions, co-expression of BiP in a bioreactor can obtain up to 8 g/L of functional scFv. Even in the shake flask culture stage, the yield of alpaca VHHs exceeds 100 mg/L. Produce more complex but still single gene-encoded forms, such as dimeric scFv-Fc antibodies in Pichia pastoris. A production level of 10-30 mg/L was achieved. Antibody formats encoded by two genes (such as Fab and IgG) require the fusion of two different antibody chains to the amino-terminal secretion signal sequence and co-transformation. In shake flask culture, the yield of Fab ranges from 1 to 50 mg/L and can reach 0.5 g/L in a bioreactor.

Antibody structure and antibody engineering design-tekbiotech.png

Figure 1 Antibody (IgG) structure and antibody engineering design

III. What are the advantages compared with other expression systems?

Prokaryotic expression systems must consider the production of antibody repertoires rather than antibody production. In addition, the higher frequency of homologous transformations in yeast compared to higher eukaryotic organisms facilitates the process of making stable expression clones. Specific issues of heterologous protein expression in yeast can be addressed by optimizing the gene sequence, for example, DNA shuffling improves the productivity of antibody fragments in yeast by avoiding AT enrichment that may lead to premature transcription termination.

In addition, yeast expression systems are inexpensive, have defined culture media, and are able to produce high cell densities of up to 100 g dry cells/L in short culture times. Currently, volumetric productivity of about 5-10 mg/L/h can be achieved by yeast culture, which exceeds most cells (~ 1-2 mg/L/h). In addition, yeast is preferred over E. coli and other bacterial hosts as an antibody production platform due to fewer glycosylation issues, higher solubility, and simpler purification processes.

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