Single Domain Antibody (sdAb) Expression in Yeast System
Yeast System for sdAb Production
In the last couple of years, yeast expression systems, especially the Saccharomyces cerevisiae and methylotrophic Pichia pastoris, have gained significant interest in the production of high levels of antibody fragments. The use of a eukaryotic expression host is a practical alternative to time-intensive optimization of bacterial expression. Antibody fragments can be expressed both intracellularly and extracellularly, i.e., secreted. The secretion pathway from yeast mostly mimics the natural expression and folding pathway of sdAbs and provides a direct route to functional sdAb fragments. Yeast system enables the secretion of soluble, functional, and correctly folded antibodies to the culture medium.
Basic Strategies for Yeast System
- sdAb Production in Pichia pastoris
The Generally Recognized as Safe (GRAS) microorganism Pichia pastoris has been one of the most successful antibody fragments expression systems. This microorganism does not accumulate toxic ethanol, nor do they secrete a large number of endogenous proteins in high-cell-density fermentation. There are two types of promoter systems available in Pichia pastoris: the tightly regulated methanol-inducible AOX1 promoter and the constitutive glyceraldehyde-phosphate dehydrogenase (GAPDH) promoter. Specifically, the methanol inducible AOX1 promoter represents the most significant advantage of Pichia pastoris and is suitable for the high expression of sdAbs.
Optimal expression in Pichia pastoris is dependent on a range of factors, including codon usage, proper aeration, temperature control (at 28-30°C), and methanol concentration; so expression cultures are grown with vigorous shaking (350 rpm). The sdAbs could be successfully expressed in Pichia pastoris with yields up to 200 mg/L in shaker flasks following the addition of an inducer and a series of optimizations. It is almost several times greater than the yields of E. coli periplasmic expression and proves the better expression ability of Pichia pastoris than the bacterial expression system.
- sdAb Production in Saccharomyces cerevisiae
Saccharomyces cerevisiae is the first yeast used to be fermented, and it has also been utilized to produce sdAbs in high performance. However, the problem of sdAbs N-glycosylation also could be occurred in this system. Before starting the cloning work into the expression vector, it is crucial to check that the sdAb gene codon usage is optimal for the Saccharomyces cerevisiae system, and no glycosylation sites are present. Even though N-glycosylation is rare in sdAbs and O-glycosylation is never observed, it is worth to make sure that these typical posttranslational modifications in yeast do not affect the folding, functionality, or activity of antibody fragments. Until now, a high yield of sdAbs in shake flasks described is produced in Saccharomyces cerevisiae, and further successful large-scale fed-batch fermentation and production are followed. The sdAb production in Saccharomyces cerevisiae fits very well with fermentation processes for both industrial applications and pharmaceutical use.
Brief Workflow for sdAb Production
- Construction of yeast expression vectors
- Transformation of recombinant plasmids to yeast strains
- Growth and induction of yeast transformants
- Purification of tagged sdAb fragments
- Quality control and sdAb binding analysis
The expression of functional sdAbs using an episomal yeast expression plasmid is under the control of the promoter. The appropriate recombinant expression plasmid is transformed into yeast by lithium acetate transformation or electroporation; the stably expressed sdAbs are secreted and extracted from the yeast culture supernatant. The sdAbs are purified in quantity for use in functionality studies and further diagnostic and therapeutic applications.
Advantages of Yeast System
Yeasts are easy to manipulate and culture and can be grown to high cell densities. The availability of strong inducible promoters such as the AOX1 promoter is a further advantage of heterologous expression. Comparable to the mammalian system, yeasts contain secretion machinery by which secretory proteins are folded and assembled in the endoplasmic reticulum. It ensures the efficient formation of antibody fragments correctly. Yeasts do not secrete many homologous proteins and endotoxins; they are generally safer and simplify the processes of downstream processing.
Case Study
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