Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

Abstract

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.

Keywords: Pichia pastoris; expression system; optimization; recombinant proteins; subunit vaccines.

Figure 1

Schematic diagram of N‐linked glycan structure in a mammalian cell, Saccharomyces cerevisiae and, Pichia pastoris. (a) N‐linked glycan structure in mammalian cells commonly generates complex terminally sialylated structures. (b) In S. cerevisiae, the N‐linked glycan structure is typically hypermannosylated (Man > 50GlcNAc2). Moreover, S. cerevisiae core oligosaccharides have terminal α‐1,3 glycan linkages. (c) N‐linked glycan structure in P. pastoris typically is of the Man8‐14GlcNAc2 type with a triantennary‐branched structure. Unlike S. cerevisiae, P. pastoris does not contain potentially immunogenic terminal α‐1,3‐linked mannoses. (d) In Pichia GlycoSwitch® strains (SuperMan5) N‐linked glycan structure is typically hypomannosylated (with a mannose‐5 structure)

Figure 2

Crossover recombination phenomenon in the Pichia pastoris genome. Following the electroporation process of competent yeast cells, cloned linear vectors are inserted into the electroporated cells. Crossover recombination occurs between 5′ promoter (5′ P_AOX1) of vector and AOX1 region of P. pastoris genome. Consequently, cloned cells with a recombinant genome are formed. AOX1, alcohol oxidase 1; TT, transcription termination region