Features of the Yeast Expression System

Yeasts are the simplest eukaryotic organisms and like bacteria are single-celled, genetically well-characterized, easy to grow and manipulate, with costs similar to those of prokaryotic expression systems. Since yeast is an eukaryote, it have an intron excision mechanism, and possesses quality control (such as protein processing, folding, and post-translational modifications) system of a typical eukaryote. Thus, it can be used for producing recombinant proteins of eukaryotes. The common yeast species used for protein expression are Pichia pastorisand the Saccharomyces cerevisiae. Pichia pastoris, normally methanol-induced and greatly engineered in recent years, is advantageous over the other species for its high-density cell culture and high protein expression levels.

Differences between Yeast and E. coli Expression systems

Yeast differs from E. coli in way of introducing foreign genes into a host cell for protein expression. E. coli cells can stably maintain the transfected plasmids/vectors, which normally carry the replication origins and replicate along with the chromosome replication. In contrast, yeast cells usually cannot stably carry foreign plasmids/vectors, as these plasmids/vectors do not carry the replication origins. Nevertheless, a foreign gene can be integrated into a yeast chromosome. The protocol of integrating a foreign gene into a yeast genome is similar to integrating a foreign gene into genome of a mammalian cell.

Advantages of Pichia pastoris expression system

1.The alcohol oxidase (AOX) promoter can be strictly regulated
2.Cell culture can reach a high density, with a mass up to 120 g/L under aerobic fermentation
3.Proteins are of post-translational modifications
4.Recombinant proteins can be produced in forms of cytoplasmic soluble protein or secreted soluble protein
5.Secreted proteins are easily purified

Factors affecting yeast protein expression

1.Choice of promoter

P. pastoris is methylotrophic yeast widely used for protein production for research and industrial applications. In this system, the gene of interest is typically cloned under the control of either the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the alcohol oxidase 1 (AOX1) promoter, depending on whether constitutive or inducible expression is desired. The GAP promoter is a strong, constitutive promoter, while the AOX1 promoter is tightly regulated, and strongly inducible by methanol. Both promoters are capable of driving very high-level gene expression, often with the protein of interest accumulating to more than 30% of total cellular soluble protein. In general, the GAP promoter is slightly stronger than the AOX1 promoter in its induced state.

2.Codon optimization for target proteins

Yeast expression systems have the codon usage bias. Therefore, genetic codons and mRNA structures need to be optimized for protein expression.

3.Transforming cytoplasmic proteins into secreted proteins

A cytoplasmic protein can be transformed into a secreted protein by adding signal peptide at its N-terminus. However, signal peptide may affect the secretion level of a protein. In addition, other factors, such as the genotype of a transformant, the method of obtaining such transformant and the intrinsic property of the protein, affect the protein yield.

AlpHelix is the sole owner of the engineered Pichia pastoris with humanized glycosylation in China

1.Yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and thereby compromises the efficacy of most therapeutic glycoproteins;

2.In 2019 and 2020, the FDA approved the nanobody medicine Caplacizumab (trade name Cablivi) and monoclonal antibody drug Eptinezumab (trade name Vyepti), respectively. Both medicines, produced by Pichia pastoris, suggest that Pichia pastoris is suitable for mass production of medicinal biologics;

3.AlpHelix owns engineered Pichia pastoris with humanized N-glycosylation. The system has a great potential in substituting conventional mammalian cells for producing therapeutic biologics and other high-value glycoproteins, with a dramatically reduced production time and cost;

AlpHelix provides tailored services, using the engineered Pichia pastoris with humanized glycosylation