Ant Protein Expression in Pichia Pastoris

Recombinant Protein Expression
REVIEW
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Recombinant Protein Expression in Pichia pastoris

James M. Cregg,1 Joan Lin Cereghino,2 Jianying Shi,3 and David R. Higgins*
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 largescale (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 microbes value and power in commercial and research labs alike. Index Entries: Pichia pastoris; Pichia methanolica; methylotrophic yeast; heterologous protein production; foreign gene expression; yeast protein expression; fermentation.
1. Introduction
Pichia pastoris has become a highly successful system for the expression of heterologous genes. Several factors have contributed to its rapid acceptance, the most important of which include:
a promoter derived from the alcohol oxidase I gene (AOX1) of P. pastoris that is uniquely suited for the controlled expression of foreign genes; the similarity of techniques needed for the molecular genetic manipulation of P. pastoris to those of Saccharomyces cerevisiae, one of the best-characterized experimental systems in modern biology;
the strong preference of P. pastoris for respiratory growth, a key physiological trait that greatly facilitates its culturing at high cell densities relative to fermentative yeasts; and a decision in 1993 by Phillips Petroleum Company continued by Research Corporation Technologies (RCT) to release the P. pastoris expression system to academic research laboratories, the consequence of which has been an explosion in the knowledge base of the system. The successful expression of more than 200 different heterologous proteins in P. pastoris has been published (Fig. 1, Table 3). A web site has been created and is maintained by the Cregg lab that lists heterologous proteins expressed in Pichia pastoris (http:// www.kgi.edu/html/ noncore/program4.htm#jc).
*Author to whom all correspondence and reprint requests should be addressed: 1Keck Graduate Institute of Applied Life Sciences, 535 North Watson Dr. Claremont, CA 91711, Email: James_Cregg@kgi.edu. 2Oregon Graduate Institute of Science and Technology, 20,000 N.W. Walker Rd. Beaverton, OR 97006, E-mail: jlcereg@bmb.ogi.edu. 3 Oregon Graduate Institute of Science and Technology, 20,000 N.W. Walker Rd. Beaverton, OR 97006, Email: jshi@bmb.ogi.edu. *Idun Pharmaceuticals,11085 N. Torrey Pines Road La Jolla, CA 92037, E mail: dhiggins@idun.com.
Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 10736085/2000/16:1/2352/$17.50
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Cregg et al.
many P. pastoris expression vectors and other useful information can be found on the Invitrogen web site (http://www.invitrogen.com). 2. A Brief History of the Pichia pastoris Expression System The ability of certain yeast species to utilize methanol as a sole source of carbon and energy was discovered approx 30 years ago by Koichi Ogata (12). Because methanol could be inexpensively synthesized from natural gas (methane), there was immediate interest in exploiting these organisms for the generation of yeast biomass or single-cell protein (SCP) to be marketed primarily as high-protein animal feed. During the 1970s, Phillips Petroleum Company (Bartlesville, OK) developed media and methods for growing P. pastoris on methanol in continuous culture at high cell densities (>130 g/L dry cell weight) (13). However, during this same period, the cost of methane increased dramatically due to the oil crisis, and the cost of soy beans (the major alternative source of animal feed protein) decreased. As a result, the SCP process was never economically competitive. In the early 1980s, Phillips Petroleum Company contracted with the Salk Institute Biotechnology/Industrial Associates, Inc. (SIBIA), a biotechnology company located in La Jolla, CA, to develop P. pastoris as a heterologous gene expression system. Researchers at SIBIA isolated the AOX1 gene (and its promoter) and developed vectors, strains, and methods for molecular genetic manipulation of P. pastoris (1419). The combination of strong regulated expression under control of the AOX1 promoter, along with the fermentation media and methods developed for the SCP process, resulted in strikingly high levels of foreign proteins in P. pastoris. In 1993, Phillips Petroleum sold its patent position with the P. pastoris expression system to RCT, the current patent holder. In addition, Phillips Petroleum licensed Invitrogen to sell components of the system to researchers worldwide, an arrangement that continues under RCT.
Fig. 1. Results of Medline search conducted on December 1, 1999, for the word Pichia in the title or abstract. * = data for 1999 do not represent a complete year.
As a yeast, P. pastoris is a single-celled microorganism that is easy to manipulate and culture. However, it is also a eukaryote and capable of many of the posttranslational modifications performed by higher eukaryotic cells, such as proteolytic processing, folding, disulfide bond formation, and glycosylation. Thus, many proteins that end up as inactive inclusion bodies in bacterial systems are produced as biologically active molecules in P. pastoris. The P. pastoris system is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from higher eukaryotes, such as insect and mammalian tissue culture cell systems, and usually gives higher expression levels. A second role played by P. pastoris in research is not directly related to its use as a protein expression system. P. pastoris serves as a useful model system to investigate certain areas of modern cell biology, including the molecular mechanisms involved in:
the import and assembly of peroxisomes; the selective autophagic degradation of peroxisomes; and the organization and function of the secretory pathway in eukaryotes.
In this review the basic aspects of the P. pastoris expression system are highlighted. Further information on the P. pastoris system can be found in the numerous reviews describing the system (111). The DNA sequence of
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3. Pichia pastoris as a Methylotrophic Yeast
P. pastoris is one of approx 30 yeast species representing two different genera (Pichia and Candida) capable of metabolizing methanol (20). The Hansenula and Torulopsis genera are now part of the Pichia and Candida genera, repsectively (Hansenula polymorpha is now Pichia angusta) (20). The methanol metabolic pathway appears to be the same in all yeasts and involves a unique set of pathway enzymes (21). The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde, generating hydrogen peroxide in the process, by the enzyme alcohol oxidase (AOX). To avoid hydrogen peroxide toxicity, this first step in methanol metabolism takes place within a specialized organelle, called the peroxisome, which sequesters toxic hydrogen peroxide away from the rest of the cell. AOX is a homo-octomer with each subunit containing one noncovalently bound FAD (flavin adenine dinucleotide) cofactor. Alcohol oxidase has a poor affinity for O2, and methylotrophic yeasts appear to compensate for this deficiency by synthesizing large amounts of the enzyme. There are two genes in P. pastoris that code for AOXAOX1 and AOX2but the AOX1 gene is responsible for the vast majority of alcohol oxidase activity in the cell (18). Expression of the AOX1 gene is tightly regulated and induced by methanol to high levels. In methanol-grown shake-flask cultures, this level is typically approx 5% of total soluble protein but can be 30% in cells fed methanol at growth limiting rates in fermentor cultures (22). Expression of the AOX1 gene is controlled at the level of transcription (12,16,18). In methanol-grown cells, approx 5% of polyA+ RNA is from the AOX1 gene, whereas, in cells grown on other carbon sources, the AOX1 message is undetectable. The regulation of the AOX1 gene is similar to the regulation of the GAL1 gene of S. cerevisiae, in that control appears to involve two mechanisms: a repression/ derepression mechanism plus an induction mechanism. However, unlike GAL1 regulation,
25
derepressing conditions (e.g., the absence of a repressing carbon source such as glucose in the medium) do not result in substantial transcription of the AOX1 gene. The presence of methanol appears to be essential to induce high levels of transcription (16).
4. Secretion of Heterologous Proteins With P. pastoris, heterologous proteins can either be expressed intracellularly or secreted into the medium. Because P. pastoris secretes only low levels of endogenous proteins and because its culture medium contains no added proteins, a secreted heterologous protein comprises the vast majority of the total protein in the medium (23,24). Thus, secretion serves as a major first step in purification, separating the foreign protein from the bulk of cellular proteins. However, the option of secretion is usually limited to foreign proteins that are normally secreted by their native hosts. Secretion requires the presence of a signal sequence on the foreign protein to target it to the secretory pathway. Although several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. The secretion signal sequence from the S. cerevisiae -factor prepro peptide has been used with the most success. 5. Common Expression Strains All P. pastoris expression strains are derivatives of NRRL-Y 11430 (Northern Regional Research Laboratories, Peoria, IL) (Table 1). Most have a mutation in the histidinol dehydrogenase gene (HIS4) to allow for selection of expression vectors containing HIS4 upon transformation (10,11,15). Other biosynthetic gene/auxotrophic mutant host marker combinations are also available but are used less frequently. All of these strains grow on complex media but require supplementation with histidine (or other appropriate nutrient) for growth on minimal media. Three types of host strains are available that vary with regard to their ability to utilize methanol due to deletions in one or both AOX genes. Strains with deleted AOX genes sometimes are
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Table 1 P. pastoris Expression Host Strains Strain Name Y-11430 GS115 KM71 MC100-3 SMD1168 SMD1165 SMD1163 Genotype his4 aox1::SARG4 his4 arg4 aox1::SARG4 aox2::Phis4 his4 arg4 pep4 his4 prb1 his4 pep4 prb1 his4 Phenotype Wild type Mut+ His Muts His Mut His Mut+ His, protease-deficient Mut+ His, protease-deficient Mut+ His, protease-deficient
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Reference NRRL* 15 16 18 10,11 10,11 10,11
*Northern Regional Research Laboratories, Peoria, IL.
better producers of a foreign protein than wildtype strains (21,23,24). These strains also require much less methanol to induce expression, which can be useful in large fermentor cultures where a large amount of methanol is sometimes considered a significant fire hazard. However, the most commonly used expression host is GS115 (his4), which is wild-type with regard to the AOX1 and AOX2 genes and grows on methanol at the wildtype rate (methanol utilization plus [Mut+] phenotype). KM71 (his4 arg4 aox1::ARG4) is a strain in which the chromosomal AOX1 gene is largely deleted and replaced with the S. cerevisiae ARG4 gene (17). As a result, this strain must rely on the much weaker AOX2 gene for AOX and grows on methanol at a slow rate [methanol utilization slow (Muts) phenotype]. With many P. pastoris expression vectors, it is possible to insert an expression cassette and simultaneously delete the AOX1 gene of a Mut+ strain (10,11,25). The third host MC100-3 (his4 arg4 aox1::SARG4 aox2::Phis4) is deleted for both AOX genes and is totally unable to grow on methanol (methanol utilization minus [Mut] phenotype) (10,11,28,26). Some secreted foreign proteins are unstable in the P. pastoris culture medium in which they are rapidly degraded by proteases. Major vacuolar proteases appear to be a significant factor in degradation, particularly in fermentor cultures, owing to the high cell density environment in combination with the lysis of a small percentage of cells. The use of host strains that are defective in these proteases has proven to help reduce degradation
in several instances (10,11). SMD1163 (his4 pep4 prb1), SMD1165 (his4 prb1), and SMD1168 (his4 pep4) are protease-deficient strains that may provide a more suitable environment for expression of certain heterologous proteins. The PEP4 gene encodes proteinase A, a vacuolar aspartyl protease required for the activation of other vacuolar proteases, such as carboxypeptidase Y and proteinase B. Proteinase B, prior to processing and activation by proteinase A, has about half the activity of the processed enzyme. The PRB1 gene codes for proteinase B. Therefore, pep4 mutants display a substantial decrease or elimination in proteinase A and carboxypeptidase Y activities, and partial reduction in proteinase B activity. In the prb1 mutant, only proteinase B activity is eliminated, whereas pep4 prb1 double mutants show a substantial reduction or elimination in all three of these protease activities.
6. Expression Vectors Plasmid vectors designed for heterologous protein expression in P. pastoris have several common features (Table 2). The foreign gene expression cassette is one of those and is composed of DNA sequences containing the P. pastoris AOX1 promoter, followed by one or more unique restriction sites for insertion of the foreign gene, followed by the transcriptional termination sequence from the P. pastoris AOX1 gene that directs efficient 3' processing and polyadenylation of the mRNAs. Many of these vectors also include the P. pastoris HIS4 gene as a selectable marker
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Vector name pHIL-D2 pAO815 Targeting Intracellular Intracellular
Table 2 Common P. pastoris Expression Vectors Selectable markers Features HIS4 HIS4 NotI sites for AOX1 gene replacment Expression cassette sites bounded by BamHI and BglII for generation of multicopy expression vector Multiple cloning sites; for insertion of foreign genes; G418 selection for multicopy strains Multiple cloning sites for insertion of foreign genes; Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags Expression controlled by constitutive GAPp Expression controlled by constitutive GAPp; multiple cloning site for insertion of foreign genes; Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags AOX1p fused to PHO1 secretion signal; XhoI, EcoRI, and BamHI sites available for insertion of foreign genes AOX1p fused to -MF prepro signal sequence; XhoI (not unique), EcoRI, NotI, SnaBI, and AvrII sites available for insertion of foreign genes; G418 selection for multicopy strains AOX1p fused to -MF prepro signal sequence; multiple cloning site for insertion of foreign genes; Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags Expression controlled by constitutive GAPp; GAPp fused to -MF prepro signal sequence; multiple cloning site for insertion of foreign genes; Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags
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References Sreekrishna, personal comm. 2
pPIC3K
Intracellular
HIS4 and kanr bler
pPICZ
Intracellular
10
pHWO10 pGAPZ
Intracellular Intracellular
HIS4 bler
29 Invitrogen, Carlsbad, CA
pHIL-S1
Secreted
HIS4
pPIC9K
Secreted
HIS4 and kanr
Sreekrishna, personal comm; Invitrogen, Carlsbad, CA 32
pPICZ
Secreted
bler
10
pGAPZ
Secreted
bler
Invitrogen, Carlsbad, CA
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for transformation into his4 mutant hosts of P. pastoris, as well as sequences required for plasmid replication and maintenance in bacteria (i.e., ColE1 replication origin and ampicillin-resistance gene). Some vectors also contain AOX1 3' flanking sequences that are derived from a region of the P. pastoris genome that lies immediately 3' of the AOX1 gene and can be used to direct fragments containing a foreign gene expression cassette to integration at the AOX1 locus by gene replacement or gene insertion 3' to AOX1 gene (10,11). Additional features that are present in certain P. pastoris expression 2vectors serve as tools for specialized functions. For secretion of foreign proteins, vectors have been constructed that contain a DNA sequence immediately following the AOX1 promoter that encodes a secretion signal. The most frequently used of these is the S. cerevisiae -factor prepro signal sequence (10,11,27,28). However, vectors containing the signal sequence derived from the P. pastoris acid phosphatase gene (PHO1) are also available. Vectors with dominant drug-resistance markers that allow for enrichment of strains that receive multiple copies of foreign gene expression cassettes during transformations have been developed. One set of vectors (pPIC3K and pPIC9K) contains the bacterial kanamycin-resistance gene and confers resistance to high levels of G418 upon strains that contain multiple copies of these vectors (10,11,28). Another set of vectors (the pPICZ series) contains the Sh ble gene from Streptoalloteichus hindustanus (10,11). This gene is small (375 bp) and confers resistance to the drug Zeocin in E. coli, yeasts (including P. pastoris), and other eukaryotes. Because the ble gene serves as the selectable marker for both E. coli and P. pastoris, the ZeoR vectors are much smaller (approx 3 kb) and easier to manipulate than other P. pastoris expression vectors. These vectors also contain a multiple cloning site (MCS) with several unique restriction sites for convenience of foreign gene insertion and sequences encoding the His6 and myc epitopes so that foreign proteins can be easily epitope-tagged at their carboxyl termini, if desired.
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Another feature present on certain vectors (e.g., pAO815 and the pPICZ vector series) is designed to facilitate the construction of expression vectors with multiple expression cassette copies (10). Multiple copies of an expression cassette are introduced in these vectors by inserting an expression cassette bounded by a BamHI and a BglII site into the BamHI site of a vector already containing a single expression cassette copy. The resulting BamHI/BglII junction between the two cassettes can no longer be cleaved by either enzyme allowing for the insertion of another BamHIBglIIbounded cassette into the same vector to generate a vector with three cassette copies. The process of addition is repeated until 68 copies of a cassette are present in a single final vector, which is then transformed into the P. pastoris host strain. Recently, vectors containing alternative promoters have become available (Table 2). Unlike the AOX1 promoter, these promoters do not require induction by methanol, which may be problematic in some instances. One is a strong constitutive promoter derived from the P. pastoris glyceraldehyde-3-phosphate dehydrogenase gene (GAP) (29). In addition to not involving methanol, the GAP promoter is convenient since cultures do not need to be shifted from one carbon source to another to induce expression of a foreign gene. However, this promoter is not suitable for the expression foreign genes whose products are toxic to P. pastoris since the constant highlevel expression can result in the selection of nonexpressing derivative strains that have either lost the expression vector or have gained mutations that reduce or eliminate expression of the foreign gene. The second promoter is from the P. pastoris formaldehyde dehydrogenase gene (FLD1) (30). FLD1 is required for growth of P. pastoris on either methanol as a sole carbon source or certain methylated amines such as methylamine as a sole nitrogen source and is strongly induced by either of these conditions. As a result, expression of a foreign gene placed under control of the FLD1 promoter is repressed in media containing glucose and ammonium ions but can be independently induced using media containing either methanol
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or methylamine. With the FLD1 promoter, only one expression vector and strain need be constructed to examine expression under both these conditions. Importantly, both the GAP and FLD1 promoters express foreign genes at levels comparable to those observed with the AOX1 promoter.
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any additional vector sequences. These events account for 1050% of His+ transformant colonies and appear to occur at highest frequency when using electroporation to introduce vector DNAs. Multiple gene insertion events at a single locus occur spontaneously at a low but detectable frequencybetween 1 and 10% of His+ transformants (10,11,31,32). Multicopy events can occur as gene insertions either at the AOX1 or his4 loci and can be detected by DNA analysis methods (e.g., PCR, Southern/dot blotting, or differential hybridization) (3234) or by methods that directly examine levels of the foreign protein (e.g., activity assay, SDS-PAGE, or colony immunoblotting) (31,32,35). It is possible to enrich transformant populations for ones that have multiple copies of an expression vector by use of either a G418R or ZeoR gene-containing vector and selecting for hyper-resistance to the appropriate drug (10,11,28,32). It is important to note that, with the G418R vectors, it is essential to first select for His+ transformants and to then screen for ones that are resistant to G418R (32). With ZeoR vectors, it is possible to directly select for hyper-Zeo-resistant transformants (10). Drug-resistant strains resulting from either the G418R or ZeoR selection methods can contain between one and five copies of the expression vector. To find strains with 20 or more copies, it is usually necessary to screen more than 100 drug-resistant strains.
7. Integration of Vectors into the Pichia pastoris Genome As in S. cerevisiae, linear vector DNAs can generate stable transformants of P. pastoris via homologous recombination between sequences shared by the vector and host genome (10,11,15,25). Such integrants show strong stability in the absence of selective pressure even when present as multiple copies. All P. pastoris expression vectors carry at least one P. pastoris DNA segment (the promoter fragment) with unique restriction sites that can be cleaved and used to direct the vector to integrate into the host genome by a single crossover type insertion event. Vectors containing the P. pastoris HIS4 gene can also be directed to integrate into the P. pastoris genomic his4 locus. Expression vectors that contain 3' AOX1 sequences can be integrated into the P. pastoris genome by a single crossover event at either AOX1 or HIS4 loci or by a gene replacement ( insertion) event at AOX1. The latter event arises from crossovers at both the AOX1 promoter and 3' AOX1 regions of the vector and genome, and results in the deletion of the AOX1 coding region (i.e., gene replacement). Transformants resulting from such an AOX1 replacement event are phenotypically His+ and Muts. Muts strains sometimes express higher levels of foreign protein (10). In addition, a Muts phenotype serves as a convenient indicator to confirm the presence of an integrated expression cassette in the P. pastoris genome. With either single crossover or gene replacement integration strategies and selection for His+ transformants, a significant percentage of transformants will not contain the expression vector. This appears to be due to gene conversion events between the HIS4 gene on the vector and the P. pastoris his4 locus such that the wild-type HIS4 gene recombines into the genome without
8. Posttranslational Modifications P. pastoris has the potential to perform many of the posttranslational modifications typically associated with higher eukaryotes. These include processing of signal sequences (both pre- and prepro-type), folding, disulfide bridge formation (10,11,36), and O- and N-linked glycosylation. Glycosylation of secreted foreign (higher) eukaryotic proteins by P. pastoris and other fungi can be problematic. In mammals, O-linked oligosaccharides are composed of a variety of sugars including N-acetylgalactosamine, galactose, and sialic acid. In contrast, lower eukaryotes, including P. pastoris, add O-oligosaccharides solely composed of mannose (Man) residues
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(4,10,11). The number of Man residues per chain, their manner of linkage, and the frequency and specificity of O-glycosylation in P. pastoris have yet to be determined. One should not assume that, because a protein is not O-glycosylated by its native host, P. pastoris will not glycosylate it. P. pastoris added O-linked mannose to approx 15% of human IGF-1 protein, although this protein is not glycosylated at all in humans. Furthermore, one should not assume that the specific Ser and Thr residue(s) selected for O-glycosylation by P. pastoris will be the same as the native host. N-glycosylation in P. pastoris and other fungi is also different than in higher eukaryotes (4,10,11). In all eukaryotes, it begins in the endoplasmic reticulum with the transfer of a lipid-linked oligosaccharide unit (Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = Nacetylglucosamine) to asparagine at the recognition sequence Asn-X-Ser/Thr. This oligosaccharide core unit is subsequently trimmed to Man8GlcNAc2. It is at this point that lower and higher eukaryotic glycosylation patterns begin to differ. The mammalian Golgi apparatus performs a series of trimming and addition reactions that generates oligosaccharides composed of either Man5 6GlcNAc2 (high-mannose type), a mixture of several different sugars (complex type), or a combination of both (hybrid type). Two distinct patterns of N-glycosylation have been observed on foreign proteins secreted by P. pastoris. Some proteins, such as S. cerevisiae invertase, are secreted with carbohydrate structures similar in size and structure to the core unit (Man811GlcNAc2) (26,36). Other foreign proteins secreted from P. pastoris receive much more carbohydrate and appear by SDS-PAGE and Western blotting to be hyperglycosylated (10,11,23). Interestingly, P. pastoris does not appear to be capable of adding 1,3-terminal mannose to oligosaccharides (R. Trimble, personal communication). This contrasts with S. cerevisiae oligosaccharides where 1,3linked terminal mannose is common. Aside from the probable absence of 1,3-linked mannose, little is known regarding the structure of P. pastoris outer-chain oligosaccharides. Furthermore, it is also not clear why outer chains are added to some
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P. pastoris-secreted proteins and not others, nor how outer chain addition may be prevented. N-linked high-mannose oligosaccharides added to proteins by yeasts represent a significant problem in the use of foreign-secreted proteins by the pharmaceutical industry. They can be exceedingly antigenic when introduced intravenously into mammals and are rapidly cleared from the blood by the liver. An additional problem caused by the differences between yeast and mammalian N-linked glycosylation patterns is that the long outer chains can potentially interfere with the folding or function of a foreign protein.
9. Expression in Fermentor Cultures Although a few foreign proteins have expressed well in P. pastoris shake-flask cultures, expression levels in shake-flasks are typically low relative to what is obtainable in fermenter cultures. One reason fermenter culturing is necessary is that only in the controlled environment of a fermenter is it possible to grow the organism to high cell densities (>100 g/L dry cell weight or 500 OD600 units/mL). Especially for secreted proteins, the concentration of product in the medium is roughly proportional to the concentration of cells in culture. A second reason is that the level of transcription initiated from the AOX1 promoter can be 35 times greater in P. pastoris cells fed methanol at growth-limiting rates in fermenter culture relative to cells grown in excess methanol. Thus, even for intracellularly expressed proteins, yields of product from a given strain as a percentage of total cellular proteins are significantly higher from fermenter cultured cells. A third reason is that methanol metabolism utilizes oxygen at a high rate, and expression of foreign genes is negatively affected by oxygen limitation. Only in the controlled environment of a fermenter is it feasible to accurately monitor and adjust oxygen levels in the culture medium. Thus, most users of the P. pastoris expression system should expect to produce their foreign protein in fermenters. A hallmark of the P. pastoris system is the ease by which expression strains scale up from shakeflask to high-density fermenter cultures. Considerable effort has gone into the optimization of high-cell-density fermentation techniques for
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Table 3 Heterologous Proteins Expressed in Pichia.pastoris Comments Mode, amount, signal sequence S, 2.5 g/L, SUC2 S, 100 mg/L, native I, 3 g/L I, 78 mg/L I, 390 g/g I, 2.4 mg total I, 12 g/L S, 28.9 U/mg I, 2.0 103 U/mg I S, -MF S, 50 mg/L, -MF I, 77 mg/L S S, 2.47 g/L total protein, -MF S, 12 mg/L, -MF S, 7 mg/L, -MF
31
Protein
References 53, 54 55 34 56 57 58 33 59 16 29 60 61 62 63 64 65 66
Bacteria
Bacillus licheniformis -amylase Bacillus stearothermophilus D -alanine carboxypeptidase Bordetella pertussis pertussis pertactin (P69) Clostridium botulinum neurotoxin (BoNT) serotype A and B Clostridium botulinum neurotoxin heavy chain fragment, serotype B Clostridium botulinum neurotoxin serotype A binding domain Clostridium tetani tetanus toxin fragment C Escherichia coli acid phosphatase/phytase (appA2) Escherichia coli -galactosidase Escherichia coli -lactamase Leishmania major cathepsin B-like protease Staphylococcus aureus staphylokinase Streptococcus equisimili streptokinase Streptomyces subtilisin inhibitor Streptomyces viridosporus T7A peroxidase, endoglucanase Toxoplasma gondii SAG1 antigen Vibrio cholerae accessory cholera enterotoxin (Acc)
Fungi
Alternaria Alt 1 allergen Aspergillus awamori glucoamylase Aspergillus awamori glucoamylase catalytic domain Aspergillus fumigatus catalase L Aspergillus fumigatus dipeptidyl peptidase IV (DPP IV) Aspergillus fumigatus dipeptidyl peptidase V (DPP V) Aspergillus giganteus -sarcin ribotoxin Aspergillus niger phytase (phyA) Candida guilliermondii xylose reductase gene (xylI) Candida rugosa lipase 1 (CRL) Fusarium solani pectate lyase (pelC) Fusarium solani pectate lyase (pelD) Geotrichum candidum lipases isoenzymes Phytophthora cryptogea -cyptogein Rhizopus oryzae lipase Saccharomyces cerevisiae invertase Saccharomyces cerevisiae Ktr1p Saccharomyces cerevisiae S, -MF S, 400 mg/L, native S, 400 mg/L, PHO1 S, 2.3 g/L, PHO1 S, PHO1 S, 0.15 mg/L, PHO1 S, 1 mg/L, synthetic native, PHO1 S, 65 U/ml, -MF I, 0.65 U/mg; S, 0.18 U/mg, -MF S, 150 U/ml, -MF S, 1 mg/L, PHO1 S, native S, 60 mg/L, -MF S, 45 mg/L, PHO1 S, 60 mg/L, -MF S, 2.5 g/L, native S, 400 mg/L, PHO1 S, 40 mg/L, PHO1 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 23 82 82
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(-1,2-mannosyltransferase) Schizophyllum commune vitamin B2aldehyde-forming enzyme Trametes versicolor (white rot fungus) laccase (lccI) Trichoderma harzianum -(1-6)-glucanase
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S, 120 mg/L, -MF S, native and -MF S, 9.3 mg/L I I S, 24 mg/L, -MF S, 50 mg/L, PHO1 I, 400 g/g S, 11.5 g/L, native I, 2.167 U/g I, 18 g/g S, -MF S, 50 mg/L, native S, 1 mg/L, native S, -MF S, 400 mg/L, -MF S, 1 mg/L, native S, 1.5 g/L, PHO1 S, PHA-E I, 22 g/L S, 10 mg/L, -MF S, native I, 30 g/g I, 20 g/g S, 60 mg/L, -MF S, -MF S, native S, 50 mg/L, -MF S, native S, -MF I, 25 g/g S, 1 mg/L, -MF S, 10 mg/L, -MF I, 250 U/g I, 0.5 mg/g S, PHO1 S, -MF S, PHO1 S, 720 mg/L, PHO1
83 84 85 86 87 40 88 89 90 91 92, 93 94 95 95 96 97 98 99, 100 101 102 103, 104 105 106, 107 108 109 110 110 111 101 112 113 114 115 116, 117 118 119 120 121 122
Protists
Chondrus crispus red alga hexose oxidase Gracilariopsis lemaneiformis red alga -1,4-glucan lyase (GLq1) Plasmodium falciparum merozoite surface protein 1 (MSP-1) Plasmodium vivax apical Membrane antigen I (AMA-1) Reticulomyxa filosa (giant freshwater amoeba) 2, 2 tubulin isoforms Trypanosoma cruzi acid -mannosidase
Plants
Allium sativum (garlic) alliin lyase Arabidopsis thaliana NADH:nitrate reductase Barley (Hordeum vulgare) sucrose fructan 6-fructosyl transferase Barley -amylase 1 Barley -amylase 2 Barley aleurone tissue -glucosidase Coffee bean -galactosidase Cynara cardunculus (cardoon) cyprosin Cynodon dactylon (Bermuda grass) Cyn d 1 Galanthus nivalis agglutinin Hevea brasiliensis hydroxynitrile lyase Hevea brasiliensis Hev b 7 patatin-like allergen Maize cytokinin oxidase Oat phytochrome A, phA Oat phytochrome A, phyA65 apoprotein Olea europaea (olive tree) aeroallergen Ole e 1 Pepper endo--1,4-glucanase cCel1 Pepper endo--1,4-glucanase cCel2 Persea americana (avocado) prs a 1 major allergen Phaseolus vulgaris agglutinin (phytohaemagglutinin) Phleum pratense -expansin Potato phytochrome B Ragweed allergen Amb a 6 Soybean root nodule acid phosphatase Spinach glycolate oxidase Spinach phosphoribulokinase Sugar beet defensin AX2 Timothy grass group I allergen Tomato Lycopesicon esculentum Mill. LeMir (L. esculentum miraculin) Wheat lipid transfer protein
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Invertebrates
Achacina fulica Ferussac (giant African snail) achacin Aplysia californica (marine invertebrate) ADP ribosyl cyclase Aqueora victoria (jellyfish) green fluorescent protein Boophilus microplus (cattle tick) Bm86 Cockroach allergen, Bla g 4 Drosophila melanogaster angiotensin I-converting enzyme Drosophila melanogaster caritine palmitoyltransferase Firefly luciferase GAVAC vaccine against cattle tick Haementeria ghilanii (South American leech) ghilanten Hirudo medicinalis (leech) hirudin Honeybee odorant-binding protein (ASP2) Honeybee olfactory protein Nippostrongylus brasiliensis (parasitic nematode) nonneuronal secreted acetylcholine sterase Spider dragline silk protein Tick anticoagulant peptide Yellowjacket venom allergen, Ag5 S, 0.2 mg/L, native S, 300 mg/L, -MF I, S, PHA-E I, S*, 1.5 g/L, SUC2 S, 50 mg/L S, 160 mg/L, -MF I (mitochondria) I (peroxisome) S, 2.0 g/L S, 10 mg/L, -MF S, 1.5 g/L, -MF S, 150 mg/L, native S, 0.2 g/L, native S, 27 mg/L, -MF I, 663 mg/L S, 1.7 g/L S, 100 mg/L, -MF S, 6.3 mg/L, -MF S, 4 g/ml, -MF S, 4 mg/L, SUC2 S, 550 mg/L, native S*, 0.3 mg/L, PHO1 S, 930 mg/L, -MF I, 1 g/L S, >1 g/L, -MF S, 1.1 mg/L, -MF S, -MF S, -MF S, 2 mg/L, native S, 11.6 mg/L, -MF, PHO1 S, native S, 20 mg/L, -MF S, mg amounts, native S*, 40 pmol/mg, -MF S, 450 mg/L, -MF S, 14.8 g/l, -MF S, 10 mg/L, -MF S, native S, 270 mg/L, native I (membrane-bound), 6 g/mg S, 250 mg/L, a-MF, PHO1 140 27 141 142 143 144 145 146 147 148 4244 149 150 151 152 153 154 45 155 156 28 157 158 159 160 161163 164 123 124 101,125 126129 130 131 132 133 134 135 136 137 138 139
33
Vertebrates (nonhuman)
Bovine enterokinase catalytic domain Bovine follicle-stimulating hormone -subunit Bovine IFN- 1 Bovine lysozyme c2 Bovine opsin Bovine pancreatic trypsin inhibitor (aprotinin) Bovine -casein Bovine -lactoglobulin Bovine tissue-type plasminogen activator (tPA) Bovine transcobalamin Brushtail possum TNF Bungarus fasciatus (snake) venom gland acetylcholinesterase Chicken liver -N-acetylgalactosaminidase Electrophorus electricus acetylcholinesterase AChE type T Hen lysozyme Mammalian lipocalin allergen Bos d2 Mouse 5HT5A 5-tryptamine receptor Mouse epidermal growth factor Mouse gelatin Mouse gelatinase B Mouse lysosomal acid -mannosidase Mouse major urinary protein complex (MUP) Mouse Mdr3 P-glycoprotein Mouse single-chain Fv fragments (sFv)
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Murine endostatin Murine Golgi mannosidase IA Murine macrophage inflammatory protein-2 (MIP-2) Ovine follicle-stimulating hormone (oFSH) Porcine carboxypeptidase B Porcine follicle-stimulating hormone Porcine inhibitor of carbonic anhydrase (transferrin family) Porcine leukocyte 12-lipoxygenase Rabbit intestinal peptide transporter (PEPT1) Rabbit intestinal peptide transporter (PEPT2) Rabbit monoclonal single-chain Fv specific for recombinant human leukemia inhibitory factor Rabbit plasma cholesteryl ester transfer protein Rabbit testicular angiotensin-converting enzyme Rat acetycholinesterase Rat brain acetylcholinesterase T subunit Rat complement regulator, crry Rat Golgi sialoglycoprotein MG160 Rat high-mobility group 1 (HMG 1) Rat liver mitochondrial carnitine palmitoyl transferases I and II (CPTI and II) Rat neural cell adhesion molecule Rat NO synthase reductase domain Rat peroxisomal multifunctional enzyme (perMFE-II) Rat procathepsin B Sea raven type II antifreeze protein (SRAFP) Shark 17-hydroxylase/C17,20-lyase Syrian golden hamster prion protein PrPc S, 200 mg/L, -MF S, PHO1 S, 40 mg/L, -MF S, 22 mg/L, -MF S, 200 mg/L, -MF S, 10 mg/L, PHO1 S, 5 mg/L, -MF I I I S, 100 mg/L, -MF S, PHO1 S, PHO1, native S, 1 mg/L, native S, 100 U/L -MF S, -MF S, 10 mg/L, -MF S, 50 mg/L, -MF I (mitochondria) S, -MF I, 25 mg/L I S, 100 mg/L, -MF S, 30 mg/L, -MF I I, <0.1 mg/L S, 30 mg/L, -MF S, -MF S, 11.6 mg/L, -MF S, inulinase signal sequence I S*, 25 nmol/g, -MF S*, a-MF I, 1 mg/L S, PHO1 S, 24 mg/L, 0.1 mg/L, -MF S, 40 mg/L, -MF S, 24 mg/L, PHO1 S, 4.5 + 1 mg/L, native S, 300 mg/L, native, INV S, 1 mg/L, -MF I, 0.2 mg/L S, 20 mg/L, -MF
Cregg et al.
165 166 167 168 169 170 171 172 173 174 175 176 177 152 178 179 180 181 182,183 184 185 186 187,188 37,189 190 191 126 192 193 194 195 156 196 197 198 199 200 201203 204 205 206 207,208 209
Humans
(1,3/4) Fucosyltransferase -1,2-Mannosidase 1B w/o TM domain -N-Acetylgalactosaminidase (-NAGAL) 1-Antitrypsin (1-AT) 2-Antiplasmin 2-Adrenergic receptor -Opioid receptor ADAR1, ADAR2, ds-RNA-specific adenosine deaminases Alzheimers disease amyloid precursor protein , , and -secretase products Alzheimers disease amyloid precursor protein, 2 domains Amyloid precursor-like protein 2 (APLP2) Amyloid precursor protein (APP) Amyloid precursor proteins, rAPP695, rAPP770 Bile salt-stimulated lipase Bivalent diabody against carcinoembryonic antigen (CEA), T-cell coreceptor CD2 c-Kit receptor kinase domain Carcinoembryonic antigen
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Caspase-3 Cathepsin K Cathepsin L propeptide Cathepsin V Cathepsin X CD38 CD40 ligand soluble form Chimeric B7-2 antibody fusion protein Chorionic gonadotropin subunit, subunit, and heterodimer Cromer blood group antigen decay-accelerating factor Cytomegalovirus ppUL44 antigen Decay-accelerating factor DAF (CD55)-Echovirus-7-receptor Double-stranded RNA-specific editase I (hREDI) Endostatin Erythropoietin (EPO) receptor Fas ligand Fibrinogen, 143411, 143427 Fibroblast collagenase (proMMP-1) Fibrinogen-420 EC domain Gastric cathepsin E Gelatinase B Granzyme B Heart muscle carnitine palmitoyltransferase I (M-CPTI) Insulin Insulin-like growth factor-1 (IGF-1) Interferon- receptor cytoplasmic domain 15N-Interferon Interleukin-17 (hIL-17) Intracellular proteinase inhibitor (PI-6) Kunitz-type protease inhibitor domain of protease nexin-2/amyloid -protein precursor Leukemia inhibitory factor (LIF) Lymphocyte surface antigen CD38 Lysosomal -mannosidase Mast cell tryptase MHC class II heterodimers (soluble form/HLA-DR2) Monoclonal single-chain Fv Monocyte chemoattractant protein-1 (MCP-1) Monocyte chemotactic protein-3 (hMCP-3) NEFA (DNA binding, EF-hand, Acidic amino acid rich region) Neural cell adhesion molecule (NCAM) NonO nucleic acid binding protein Oncostatin-M Pancreatic -amylase Pancreatic triglyceride lipase Papain nitrile hydratase Placental alkaline phosphatase (PLAP) I, 1 g/g S, 38 mg/L, -MF S, 10 mg/L, -MF S, -MF S, 5 mg/L, -MF S, 455 mg/L, -MF S, 255 mg/L S, 15 mg/L, -MF S, 24 mg/L (), 3mg/L (), 16 mg/L (), -MF S, -MF I, 0.1 mg/mL S, 6 mg/L, -MF I, 1 mg/L S, 20 mg/L, -MF S, 200 mg/L, PHO1 S, 100 mg/L, -MF S, 100 mg/L, 75 mg/L, -MF S, 2.3 mg/L, -MF S, -MF S, 0.6 mg/L, native S, 50 mg/L, -MF S, 1 mg/L, -MF I (mitochondria) S, synthetic signal S, 600 mg/L, -MF I S, 810 mg/L, PHO1 S, 0.35 mg/L, -MF I, 50 mg/L S, 1.0 g/L, -MF S, 17 mg/L, -MF S, 400 mg/L, PHO1 S, 83 g/L, native S, 6.5 mg/L, -MF S, 400 g/L, -MF S, 50 mg/L, -MF S, 100 mg/L, native or -MF S, 1 mg/L, PHO1 S, 1001200 mg/L, -MF S, 50 mg/L, PHO1 I (endoplasmic reticulum) S, 50 mg/L, -MF S, 20 mg/L, -MF S, 75 ml/L, PHO1 S, 5 mg/L, -MF S, 2 mg/L, PHO1 210 211,212 213,214 215 216 217 218 219 220 221 222 223 224 165,225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245,246 247 248 249 250 251 252 253 254 255 256 257 258
35
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Placental protein-14 (PP-14) Plasminogen kringles 1-4 Plasminogen kringles 1-4, angiostatin protein Plasminogen kringles K2 Procarboxypeptidase A2 Procathepsin B Procolipase Protein kinase C interacting protein-1 (PKCI-1) Proteinase 3, Wegeners antigen Proteinase inhibitor 8 scFv (against ovarian carcinoma)biotin mimetic peptide scFv (against squamous carcinoma) Serum albumin Serum transferrin N-Lobe Sex steroid binding protein Single-chain urokinase-type plasminogen activator Thrombomodulin Tissue factor extracellular domain Tissue kallikrein Tissue-type plasminogen activator kringle 2 domain Transforming growth factor receptor extracellular domain Tumor necrosis factor (TNF) Type 1 plasminogen activator inhibitor (PAI-1) Type III collagen (with prolyl 4-hydroxylase) Urokinase-type plasminogen activator-annexin V chimera
Vascular endothelial growth factor (VEGF165)
Cregg et al.
S, -MF S, 17 mg/L, PHO1 S, 10% total protein, PHO1 S, 160 mg/L, -MF S, 180 mg/L, -MF S, 20 mg/L, -MF S, 30 mg/L, native I, 0.25 mg/L S, 670 mg/L, -MF I, 15% total protein S S, 50 mg/L, -MF S, 3 g/L, native S, 240 mg/L, -MF S, 4 mg/L, -MF S, 5 mg/L, pre Mucor pusillus rennin signal S S, 10 mg/L, PHO1 S, 30 mg/L, -MF S, 170 mg/L, -MF S, 10 mg/L, -MF I, 10 g/L S, 3 mg/L, -MF I, 15 mg/L S, 600 IU/mL, pre Mucor pussils rennin
S, 40 mg/L, PHO1 S, 3 mg/ml, -MF S, 20 mg/L, -MF S, PHO1, prM virus signal sequence I, 400 mg/L I I gp120 (ENV) S, 20 mg/L, -MF I, 0.5 mg/L I, 0.8 mg/L I S, 3 mg/L, a-MF
259 260 261 262 263 264 265 266 267 268 269,270 248 24,271274 275277 278 279 280 281 282,283 18,48 284287 288 31,289 290 291 292
293 294, 295 296, 297 298 25, 299 300 301 302 303 304 305 41
Viruses
A/VICTORIA/3/75 influenza virus neuraminidase head domain Bovine herpes virus-1 glycoprotein D Dengue virus type 1 structural gene recombinant E protein Hepatitis B virus surface antigen Hepatitis B virus surface antigen-HIV gp41 epitope chimera Hepatitis E virus ORF3 Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, Polyomavirus large T antigen Reovirus 1 core protein Reovirus 1 protein Vaccinia virus complement control protein
I = Intracellular (with subcellular location), S = Secreted, S* = Secreted to plasma membrane. Amounts are highest reported for particular protein. Signal sequences: -MF (S. cerevisiae -mating factor); PHO1 (P. pastoris acid phosphatase); SUC2 (S. cerevisiae invertase).
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expression strains, and, as a result, a variety of fed-batch and continuous culture schemes are available (10,11). All schemes involve the initial growth of strains in a defined medium on glycerol. During this period, growth is rapid but heterologous gene expression is fully repressed. Upon depletion of glycerol, a transition phase is initiated in which additional glycerol is fed to cultures at a growth-limiting rate. Finally, methanol, or a mixture of glycerol and methanol, is fed to cultures to induce expression. The time of harvest, typically the peak concentration of a foreign protein, is determined empirically for each protein. High-density fermentation of P. pastoris expression strains is especially attractive for the production of secreted proteins, because their concentration in the culture medium should increase with cell density. Unfortunately, the concentrations of other cellular materials, particularly proteases, increase as well. Three strategies have proven effective in minimizing the proteolytic instability of foreign proteins secreted into the P. pastoris culture medium. One is the addition of amino acid-rich supplements, such as peptone or casamino acids, to the culture medium which appear to reduce product degradation by acting as excess substrates for one or more problem proteases (28). A second is changing the culture medium pH (28). P. pastoris is capable of growing across a relatively broad pH range from 3.0 to 7.0 which allows considerable leeway in adjusting the pH to one that is not optimal for a problem protease. A third is the use of a protease-deficient P. pastoris host strain (10,11).
37
large numbers of mutant proteins suitable for structure/function studies. The P. pastoris yeast expression system offers key advantages relative to other mammalian expression systems and to Bacculovirus. In particular, P. pastoris grows in minimal medium that can be modified for production of 15N-labeled proteins (37-41). Even triply labeled (15N, 13C, 2H) proteins have been produced in this way (42,43). In some cases, the yields of labeled proteins compete favorably with those obtained from E. coli (4446). Another advantage of the P. pastoris yeast expression system is that glycosylated proteins can be prepared in high yields and the sugars that are attached by P. pastoris are of a simple high mannose form (47,48). These sugars are easily removed by cleavage with endoglycosidase H, leaving the core GlcNac if advantage for X-ray crystallographic analysis where a homogeneous protein sample is required (4950). Indeed, crystallography of glycosylated proteins can be extremely difficult if the protein is produced in mammalian cells or in Bacculovirus because of incomplete removal of the complex sugars that these organisms attach. A comprehensive list of heterologous proteins expressed in P. pastoris can be found in Table 3 and at www.kgi.edu/html/noncore/program 4.htm#jc.
10. Expression of Protein for Structural Studies The P. pastoris yeast expression system has gained widespread use for production of proteins for structural studies. Proteins that are normally secreted or require mammalian post-translational modifications can not be produced in an active form in E. coli. For these proteins in particular, large quantities of active protein can be produced in P. pastoris. Since P. pastoris grows rapidly on inexpensive media similar to E. coli, it is feasible to quickly and cheaply synthesize and examine
11. Pichia methanolica: New Kid on the Block Recently P. methanolica, another methylotrophic species, has been developed as a heterologous expression system (51,52). The essential features of an expression system have been developed for this yeast, including, strains, expression vectors, transformation system (mostly via non-homologous recombination), a methanol-regulated promoter for recombinant protein expression, and fermentation techniques. A possible advantage over P. pastoris is in fermentation; biomass generation during fermentation can use glucose instead of glycerol and less methanol is required for the induction of recombinant protein expression. The main disadvantage of P. methanolica is that its relatively new arrival as an expression tool means that it is less well characterized, including optiVolume 16, 2000
38
mization of parameters for transformation, protein expression, and fermentation. Similar to the situation with P. pastoris in which its use is covered by patents that are owned by RCT, patents covering the use of P. methanolica are owned by ZymoGenetics, Inc. of Seattle, WA. Although use for academic research purposes is permitted, a license is required for commercial use after an initial one-year commercial evaluation period. Also like P. pastoris, P. methanolica has been licensed to Invitrogen Corp., Carlsbad, CA for distribution to academic and commercial users. P. methanolica strains, plasmids, and licensing information can be obtained from Invitrogen.
Cregg et al.
Komives, University of California, San Diego, for information on heavy atom labeled protein.
References
1. Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423488. 2. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (NY) 11, 905910. 3. Romanos, M. (1995) Advances in the use of Pichia pastoris for highlevel expression. Curr. Opin. Biotechnol. 6, 527533. 4. Cregg, J. M. (1999) Expression in the methylotrophic yeast Pichia pastoris. In Gene Expression Systems: Using Nature for the Art of Expression (Fernandez, J. M. and Hoeffler, J. P., eds.), Academic Press, San Diego, CA, pp. 157191. 5. Cregg, J. M. and Higgins, D. R. (1995) Production of foreign proteins in the yeast Pichia pastoris. Can. J. Bot. 73(Suppl. 1), S981S987. 6. Sreekrishna, K., Brankamp, R. G., Kropp, K. E., Blankenship, D. T., Tsay, J. T., Smith, P. L., Wierschke, J. D., Subramaniam, A., and Birkenberger, L. A. (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190, 5562. 7. Gellissen, G. and Hollenberg, C. P. (1997) Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactisa review. Gene 190, 8797. 8. Higgins, D. R. (1995) Overview of protein expression in Pichia pastoris. In Current Protocols in Protein Science, Supplement 2 (Wingfield, P. T., ed.), John Wiley and Sons, New York, pp. 5.7.15.7.16. 9. Sreekrishna, K. (1993) Strategies for optimizing protein expression and secretion in the methylotrophic yeast Pichia pastoris. In Industrial Microorganisms: Basic and Applied Molecular Genetics (Baltz, R. H., Hegeman, G. D., and Skatrud, P. L., ed.), American Society for Microbiology, Washington, DC, pp. 119126. 10. Higgins, D. R. and Cregg, J. M. (1998) Pichia Protocols, Methods in Molecular Biology, vol. 103, Humana Press, Totowa, NJ. 11. Cereghino, J. L. and Cregg, J. M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev., 24, 45-66. 12. Ogata, K., Nishikawa, H., and Ohsugi, M. (1969) A yeast capable of utilizing methanol. Agric. Biol. Chem. 33, 15191520. 13. Wegner, G. H. (1990) Emerging applications of the methylotrophic yeasts. FEMS Microbiol. Rev. 7, 279283.
12. Summary Based on available data, there is an approx 50 75% probability of expressing any protein of interest in P. pastoris at a reasonable level. The biggest hurdle seems to be generating initial successthat is, expressing a specific protein at any level. After success at this stage, there are welldefined parameters that can be manipulated to optimize expression, and it is often at this stage that attractive levels of expression are achieved. Although there are relatively few examples of expression of 10 g/L, there are many examples of expression in the 1 g/L range, ranking the P. pastoris expression system as one of the most productive eukaryotic expression systems available. There are also examples of proteins that have been successfully expressed in P. pastoris that were completely unsuccessful in Baculovirus or S. cerevisiae expression systems, making the P. pastoris system an important alternative to have available in the protein expression "toolbox." See Table 3 for a comprehensive list of heterologous proteins expressed in P. pastoris. Acknowledgments The preparation of this manuscript was supported by Grant DK-43698 from the National Institutes of Health and Grant ER20334 from the Department of Energy, Office of Basic Energy Sciences (to J.M.C.). We would like to thank Terrie Hadfield, Oregon Graduate Institute, for expert assistance in preparation of the manuscript, and Elizabeth
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14. Ellis, S. B., Brust, P. F., Koutz, P. J., Waters, A. F., Harpold, M. M., and Gingeras, T. R. (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol. Cell. Biol. 5, 11111121. 15. Cregg, J. M., Barringer, K. J., Hessler, A. Y., and Madden, K. R. (1985) Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5, 33763385. 16. Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A., and Gingeras, T. R. (1987) Expression of the LacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 15, 38593876. 17. Cregg, J. M. and Madden, K. R. (1987) Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris gene disruption. In Biological Research on Yeasts, vol. 2 (Stewart, G. G., Russell, I., Klein, R. D., and Hiebsch, R. R., ed.), CRC Press, Boca Raton, FL, pp. 118. 18. Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 13161323. 19. Koutz, P. J., Davis, G. R., Stillman, C., Barringer, K., Cregg, J. M., and Thill, G. (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5, 167177. 20. Kurtzman, C. P. and Robnett, C. J. (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73, 331371. 21. Veenhuis, M., van Dijken, J. P., and Harder, W. (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv. Microb. Physiol. 24, 182. 22. Couderc, R. and Baratti, J. (1980) Oxidation of methanol by the yeast Pichia pastoris: purification and properties of alcohol oxidase. Agric. Biol. Chem. 44, 22792289. 23. Tschopp, J. F., Sverlow, G., Kosson, R., Craig, W., and Grinna, L. (1987) High level secretion of glycosylated invertase in the methylotrophic yeast Pichia pastoris. Biotechnology (NY) 5, 13051308. 24. Barr, K. A., Hopkins, S. A., and Sreekrishna, K. (1992) Protocol for efficient secretion of HSA developed from Pichia pastoris. Pharm. Eng. 12, 4851. 25. Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S., Buckholz, R. G., Madden, K. R., Kellaris, P. A., Davis, G. R., Smiley, B. L., Cruze, J., Torregrossa, R., Velicelebi, G., and Thill, G. P. (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Biotechnology (NY) 5, 479485.
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26. Chirulova, V., Cregg, J. M., and Meagher, M. M. (1997) Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations. Enzyme Microb. Technol. 21, 277283. 27. Laroche, Y., Storme, V., De Muetter, J., Messens, J., and Lauwereys, M. (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast Pichia pastoris. Biotechnology (NY) 12, 11191124. 28. Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M. M., Sreekrishna, K., and Henwood, C. A. (1991) Production of mouse epidermal growth factor in yeast: highlevel secretion using Pichia pastoris strains containing multiple gene copies. Gene 105, 205212. 29. Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S. V., and Cregg, J. M. (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186, 3744. 30. Shen, S., Sulter, G., Jeffries, T. W., and Cregg, J. M. (1998) A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216, 93102. 31. Sreekrishna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P., and Parker, K. (1989) Highlevel expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry 28, 41174125. 32. Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos, M. A., and Sreekrishna, K. (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology (NY) 12, 181184. 33. Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K., and Romanos, M. A. (1991) Highlevel expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (NY) 9, 455460. 34. Romanos, M. A., Clare, J. J., Beesley, K. M., Rayment, F. B., Ballantine, S. P., Makoff, A. J., Dougan, G., Fairweather, N. F., and Charles, I. G. (1991) Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties. Vaccine 9, 901906. 35. Wung, J. L. and Gascoigne, N. R. (1996) Antibody screening for secreted proteins expressed in Pichia pastoris. Biotechniques 21, 808, 810, 812. 36. Trimble, R. B., Atkinson, P. H., Tschopp, J. F., Townsend, R. R., and Maley, F. (1991) Structure of oligosaccharides on Saccharomyces SUC2 invertase
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Recombinant Protein Expression

Reference NRRL* 15 16 18 10,11 10,11 10,11

*Northern Regional Research Laboratories, Peoria, IL.

Recombinant Protein Expression

HIS4 and kanr bler

HIS4 and kanr

Sreekrishna, personal comm; Invitrogen, Carlsbad, CA 32

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