Review
Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris

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Abstract

The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. Using this system, recent advances have been made in the quality of recombinant proteins in fermenter culture and in the quality of the protein product, namely improved secretion signals and glycosylation patterns.

Introduction

Pichia pastoris is a methylotrophic yeast that can be genetically engineered to express proteins for both basic research and industrial use [1••]. Over 400 proteins, from human endostatin to spider dragline silk protein, have been produced in this yeast [2••]. P. pastoris is uniquely suited for foreign protein expression for three key reasons: it can be easily manipulated at the molecular genetic level (e.g. gene targeting, high-frequency DNA transformation, cloning by functional complementation); it can express proteins at high levels, intracellularly or extracellularly; and it can perform many ‘higher eukaryotic’ protein modifications, such as glycosylation, disulfide-bond formation, and proteolytic processing [3]. Because of these characteristics some proteins that cannot be expressed efficiently in bacteria, Saccharomyces cerevisiae or baculovirus have been successfully produced in functionally active form in P. pastoris [4••]. The powerful genetic techniques available, coupled with its economy of use, make P. pastoris a system of choice for heterologous protein expression. In previous reviews, we have discussed molecular genetic factors affecting the construction of P. pastoris expression strains 2••., 3., 4••., 5.. In this review, we concentrate on recent advances in the production of recombinant proteins in fermenter culture as well as certain aspects affecting the quality of the protein product, namely secretion signal selection and modification of carbohydrate structures on secreted glycoproteins.

Section snippets

Conceptual basis for the P. pastoris expression system

There are many reasons for the popularity of the P. pastoris expression system, but two are most compelling. The first is an unusually efficient and tightly regulated promoter from the alcohol oxidase I gene (AOX1) that is used to drive the expression of the foreign gene [5]. The AOX1 promoter is strongly repressed in cells grown on glucose and most other carbon sources, but is induced over 1000-fold when cells are shifted to a medium containing methanol as a sole carbon source. The ability to

High-level expression in fermenter cultures

Although expression of heterologous proteins can be done in shake-flask culture, protein levels are typically much higher in fermenter cultures. Because the mineral media for P. pastoris (containing only glycerol or methanol, biotin, salts and trace elements) are economical and well defined, this yeast is nearly ideal for large-scale production of heterologous proteins in fermenters. Most importantly, it is only in fermenters, where parameters such as pH, aeration and carbon source feed rate

Protein-specific adjustments to improve yield

The culture media described for high cell density fermenter growth and induction of recombinant P. pastoris strains are essentially unchanged from those originally developed by Phillips Petroleum Company in the 1970s for mass production of single-cell protein [1••]. For recombinant protein synthesis in fermenters, the basal salts medium along with its companion trace salts solution (PTS1) are most commonly used as the medium for both growth and induction. Although these single-cell protein

Glycosylation of recombinant proteins

P. pastoris is able to add both O-linked and N-linked carbohydrate moieties to secreted proteins. Differences in the number and type of sugar units added by humans as compared with P. pastoris pose a large problem for the use of yeast-secreted glycoproteins as therapeutic products. The yeast-secreted proteins can be extremely antigenic if introduced into the bloodstream of mammals and are rapidly cleared from the bloodstream.

Two strategies have been used to address this challenge: to change the

Secretion signals

A valuable option in this host is the availability of secretion signals that can be attached to the protein of interest, causing it to be exported out of the cell. Although a variety of secretion signal sequences have been used with success (including signals native to the foreign protein in several cases), the 85 amino acid S. cerevisiae MATα prepro signal peptide has found most success and is the one most frequently incorporated into P. pastoris expression vectors [2••]. In most cases, this

Conclusions

The P. pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. Thus, it is a suitable host for the recombinant synthesis of the millions of proteins encoded by diverse organisms on our planet. As more is learned about this organism, its use as an expression system will become increasingly more efficient and user-friendly.

Acknowledgements

The writing of this manuscript was supported by a US National Institutes of Health grant (DK-43698) and US Department of Energy grant (DE-FG03-99ER15088) to JMC.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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