Heterologous protein expression in the methylotrophic yeast Pichia pastoris
TL;DR: This paper reviews the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced and describes new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.
Abstract: During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.
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TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed.
Abstract: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.
1,237 citations
Cites background or methods from "Heterologous protein expression in ..."
...pastoris expression system is to obtain maximum amounts of expressed foreign proteins [18]....
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...The use of protease-deficient strains, such as SMD1163 (his4 pep4 prb1 ), SMD1165 (his4 pep4 ), and SMD1168 (his4 pep4 ), has been found to enhance the yield and the quality of various heterologous proteins [18]....
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...More information about the expression vectors available for this system can be found in Cereghino and Cregg [18], Cregg et al....
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...The Mut+, or methanol utilization plus phenotype, grow on methanol at the wild-type rate and require high feeding rates of methanol in large-scale fermentations [18]....
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...pastoris, a relatively high concentration of these vacuolar proteases is present in the culture broth, resulting from the combination of high cell densities and lysis of a small percentage of cells [18]....
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TL;DR: Major advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe’s value and power in commercial and research labs alike.
Abstract: The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.
927 citations
TL;DR: The purpose of this review is to summarize important developments and features of this expression system and to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein.
Abstract: The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and muts phenotypes, intracellular expression and folding pathways is examined. The roles of pre–pro signal sequences such as the alpha mating factor (α-MF) and the Glu–Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed. Copyright © 2004 John Wiley & Sons, Ltd.
804 citations
TL;DR: This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains.
Abstract: Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.
749 citations
Cites background from "Heterologous protein expression in ..."
...Expression levels from PGAP drop to about one half on glycerol and to one third when cells are grown on methanol (Cereghino and Cregg 2000)....
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...The classical P. pastoris expression system has been extensively reviewed over the years (Cereghino and Cregg 2000; Daly and Hearn 2005; Gasser et al. 2013; Jin et al. 2006; Macauley-Patrick et al. 2005)....
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TL;DR: The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels.
603 citations
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TL;DR: A gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells, was cloned and sequenced in this paper.
647 citations
TL;DR: A methylotrophic yeast, Pichia pastoris, is developed as a host for DNA transformations based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase.
Abstract: We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.
569 citations
"Heterologous protein expression in ..." refers background in this paper
...cerevisiae, and the Sh ble gene from Streptoalloteichus hindustanus which confers resistance to the bleomycin-related drug zeocin [11,26,27]....
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...Auxotrophic strains Y-11430 wild-type NRRLa GS115 his4 [11] GS190 arg4 [16] JC220 ade1 [16] JC254 ura3 [16] GS200 arg4 his4 [11] JC227 ade1 arg4 [29] JC304 ade1 his4 [29] JC305 ade1 ura3 [29] JC306 arg4 ura3 [29] JC307 his4 ura3 [29] JC300 ade1 arg4 his4 [29] JC301 ade1 his4 ura3 [29] JC302 ade1 arg4 ura3 [29] JC303 arg4 his4 ura3 [29]...
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TL;DR: Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor.
Abstract: Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.
546 citations
"Heterologous protein expression in ..." refers background in this paper
...This is rapidly followed by cleavage of Glu-Ala repeats by the Ste13 protein [41]....
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TL;DR: In this paper, the authors reported the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris.
405 citations