recombinant protein expression in pichia pastoris.pdf

Recombinant Protein Expression 23

MOLECULAR BIOTECHNOLOGY Volume 16, 2000

REVIEW

23

Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/16:1/23–52/$17.50

*Author to whom all correspondence and reprint requests should be addressed: 1Keck Graduate Institute of Applied Life Sciences, 535 NorthWatson Dr. Claremont, CA 91711, E–mail: [email protected]. 2Oregon Graduate Institute of Science and Technology, 20,000 N.W.Walker Rd. Beaverton, OR 97006, E-mail: [email protected]. 3Oregon Graduate Institute of Science and Technology, 20,000 N.W.Walker Rd. Beaverton, OR 97006, E–mail: [email protected]. *Idun Pharmaceuticals,11085 N. Torrey Pines Road La Jolla, CA 92037, E–mail: [email protected].

Recombinant Protein Expression in Pichia pastoris

James M. Cregg,1 Joan Lin Cereghino,2 Jianying Shi,3 and David R. Higgins*

Abstract

Index Entries: Pichia pastoris; Pichia methanolica; methylotrophic yeast; heterologous protein pro-duction; foreign gene expression; yeast protein expression; fermentation.

1. Introduction

Pichia pastoris has become a highly success-ful system for the expression of heterologousgenes. Several factors have contributed to itsrapid acceptance, the most important of whichinclude:

• a promoter derived from the alcohol oxidase Igene (AOX1) of P. pastoris that is uniquelysuited for the controlled expression of foreigngenes;

• the similarity of techniques needed for themolecular genetic manipulation of P. pastoristo those of Saccharomyces cerevisiae, one ofthe best-characterized experimental systems inmodern biology;

• the strong preference of P. pastoris for respira-tory growth, a key physiological trait thatgreatly facilitates its culturing at high cell den-sities relative to fermentative yeasts; and

• a decision in 1993 by Phillips Petroleum Com-pany continued by Research Corporation Tech-nologies (RCT) to release the P. pastorisexpression system to academic research labora-tories, the consequence of which has been anexplosion in the knowledge base of the system.The successful expression of more than 200 dif-ferent heterologous proteins in P. pastoris hasbeen published (Fig. 1, Table 3). A web site hasbeen created and is maintained by the Cregg labthat lists heterologous proteins expressed inPichia pastoris (http://www.kgi.edu/html/noncore/program4.htm#jc).

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology forthe 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 programto convert abundant methanol to a protein source for animal feed has been developed into what is today twoimportant biological tools: a model eukaryote used in cell biology research and a recombinant proteinproduction system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Signifi-cant advances in the development of new strains and vectors, improved techniques, and the commercialavailability of these tools coupled with a better understanding of the biology of Pichia species have led tothis microbe’s value and power in commercial and research labs alike.

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As a yeast, P. pastoris is a single-celledmicroorganism that is easy to manipulate andculture. However, it is also a eukaryote andcapable of many of the posttranslational modi-fications performed by higher eukaryotic cells,such as proteolytic processing, folding, disul-fide bond formation, and glycosylation. Thus,many proteins that end up as inactive inclusionbodies in bacterial systems are produced as bio-logically active molecules in P. pastoris. TheP. pastoris system is also generally regarded asbeing faster, easier, and less expensive to usethan expression systems derived from highereukaryotes, such as insect and mammalian tis-sue culture cell systems, and usually giveshigher expression levels.

A second role played by P. pastoris inresearch is not directly related to its use as aprotein expression system. P. pastoris serves asa useful model system to investigate certainareas of modern cell biology, including themolecular mechanisms involved in:

• the import and assembly of peroxisomes;• the selective autophagic degradation of per-

oxisomes; and• the organization and function of the secre-

tory pathway in eukaryotes.

In this review the basic aspects of the P.pastoris expression system are highlighted.Further information on the P. pastoris systemcan be found in the numerous reviews describ-ing the system (1–11). The DNA sequence of

many P. pastoris expression vectors and otheruseful information can be found on the Invitrogenweb site (http://www.invitrogen.com).

2. A Brief History of the Pichia pastorisExpression System

The ability of certain yeast species to utilizemethanol as a sole source of carbon and energywas discovered approx 30 years ago by KoichiOgata (12). Because methanol could be inex-pensively synthesized from natural gas (meth-ane), there was immediate interest in exploitingthese organisms for the generation of yeast bio-mass or single-cell protein (SCP) to be mar-keted primarily as high-protein animal feed.During the 1970s, Phillips Petroleum Company(Bartlesville, OK) developed media and meth-ods for growing P. pastoris on methanol incontinuous culture at high cell densities (>130g/L dry cell weight) (13). However, duringthis same period, the cost of methaneincreased dramatically due to the oil crisis,and the cost of soy beans (the major alterna-tive source of animal feed protein) decreased.As a result, the SCP process was never eco-nomically competitive.

In the early 1980s, Phillips Petroleum Com-pany contracted with the Salk Institute Biotech-nology/Industrial Associates, Inc. (SIBIA), abiotechnology company located in La Jolla,CA, to develop P. pastoris as a heterologousgene expression system. Researchers at SIBIAisolated the AOX1 gene (and its promoter) anddeveloped vectors, strains, and methods formolecular genetic manipulation of P. pastoris(14–19). The combination of strong regulatedexpression under control of the AOX1 pro-moter, along with the fermentation media andmethods developed for the SCP process,resulted in strikingly high levels of foreign pro-teins in P. pastoris. In 1993, Phillips Petroleumsold its patent position with the P. pastorisexpression system to RCT, the current patentholder. In addition, Phillips Petroleum licensedInvitrogen to sell components of the system toresearchers worldwide, an arrangement thatcontinues under RCT.

Fig. 1. Results of Medline search conducted onDecember 1, 1999, for the word “Pichia” in the title orabstract. * = data for 1999 do not represent a completeyear.



3. Pichia pastoris as a Methylotrophic Yeast

P. pastoris is one of approx 30 yeast speciesrepresenting two different genera (Pichia andCandida) capable of metabolizing methanol (20).The Hansenula and Torulopsis genera are nowpart of the Pichia and Candida genera,repsectively (Hansenula polymorpha is nowPichia angusta) (20). The methanol metabolicpathway appears to be the same in all yeasts andinvolves a unique set of pathway enzymes (21).The first step in the metabolism of methanol isthe oxidation of methanol to formaldehyde, gen-erating hydrogen peroxide in the process, by theenzyme alcohol oxidase (AOX). To avoid hydro-gen peroxide toxicity, this first step in methanolmetabolism takes place within a specializedorganelle, called the peroxisome, which seques-ters toxic hydrogen peroxide away from the restof the cell. AOX is a homo-octomer with eachsubunit containing one noncovalently bound FAD(flavin adenine dinucleotide) cofactor. Alcoholoxidase has a poor affinity for O2, andmethylotrophic yeasts appear to compensate forthis deficiency by synthesizing large amounts ofthe enzyme.

There are two genes in P. pastoris that code forAOX—AOX1 and AOX2—but the AOX1 gene isresponsible for the vast majority of alcohol oxi-dase activity in the cell (18). Expression of theAOX1 gene is tightly regulated and induced bymethanol to high levels. In methanol-grownshake-flask cultures, this level is typically approx5% of total soluble protein but can be ≥30% incells fed methanol at growth limiting rates in fer-mentor cultures (22). Expression of the AOX1gene 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 AOX1message is undetectable. The regulation of theAOX1 gene is similar to the regulation of theGAL1 gene of S. cerevisiae, in that controlappears to involve two mechanisms: a repression/derepression mechanism plus an inductionmechanism. However, unlike GAL1 regulation,

derepressing conditions (e.g., the absence of arepressing carbon source such as glucose in themedium) do not result in substantial transcriptionof the AOX1 gene. The presence of methanolappears to be essential to induce high levels oftranscription (16).

4. Secretion of Heterologous ProteinsWith P. pastoris, heterologous proteins can

either be expressed intracellularly or secreted intothe medium. Because P. pastoris secretes onlylow levels of endogenous proteins and because itsculture medium contains no added proteins, asecreted heterologous protein comprises the vastmajority of the total protein in the medium(23,24). Thus, secretion serves as a major firststep in purification, separating the foreign proteinfrom the bulk of cellular proteins. However, theoption of secretion is usually limited to foreignproteins that are normally secreted by their nativehosts. Secretion requires the presence of a signalsequence on the foreign protein to target it to thesecretory pathway. Although several differentsecretion signal sequences have been used suc-cessfully, including the native secretion signalpresent on some heterologous proteins, successhas been variable. The secretion signal sequencefrom the S. cerevisiae α-factor prepro peptide hasbeen used with the most success.

5. Common Expression StrainsAll P. pastoris expression strains are deriva-

tives of NRRL-Y 11430 (Northern Regional Re-search Laboratories, Peoria, IL) (Table 1). Mosthave a mutation in the histidinol dehydrogenasegene (HIS4) to allow for selection of expressionvectors containing HIS4 upon transformation(10,11,15). Other biosynthetic gene/auxotrophicmutant host marker combinations are also avail-able but are used less frequently. All of thesestrains grow on complex media but requiresupplementation with histidine (or other appropri-ate nutrient) for growth on minimal media.

Three types of host strains are available thatvary with regard to their ability to utilize metha-nol due to deletions in one or both AOX genes.Strains with deleted AOX genes sometimes are

26 Cregg et al.


better producers of a foreign protein than wild-type strains (21,23,24). These strains also requiremuch less methanol to induce expression, whichcan be useful in large fermentor cultures where alarge amount of methanol is sometimes consid-ered a significant fire hazard. However, the mostcommonly used expression host is GS115 (his4),which is wild-type with regard to the AOX1 andAOX2 genes and grows on methanol at the wild-type rate (methanol utilization plus [Mut+] phe-notype). KM71 (his4 arg4 aox1∆::ARG4) is astrain in which the chromosomal AOX1 gene islargely deleted and replaced with the S. cerevisiaeARG4 gene (17). As a result, this strain must relyon the much weaker AOX2 gene for AOX andgrows on methanol at a slow rate [methanol utili-zation slow (Muts) phenotype]. With many P.pastoris expression vectors, it is possible to insertan expression cassette and simultaneously deletethe AOX1 gene of a Mut+ strain (10,11,25). Thethird host MC100-3 (his4 arg4 aox1∆::SARG4aox2∆::Phis4) is deleted for both AOX genes andis totally unable to grow on methanol (methanolutilization minus [Mut–] phenotype) (10,11,28,26).

Some secreted foreign proteins are unstable inthe P. pastoris culture medium in which they arerapidly degraded by proteases. Major vacuolarproteases appear to be a significant factor in deg-radation, particularly in fermentor cultures, owingto the high cell density environment in combina-tion with the lysis of a small percentage of cells.The use of host strains that are defective in theseproteases has proven to help reduce degradation

in several instances (10,11). SMD1163 (his4 pep4prb1), SMD1165 (his4 prb1), and SMD1168(his4 pep4) are protease-deficient strains that mayprovide a more suitable environment for expres-sion of certain heterologous proteins. The PEP4gene encodes proteinase A, a vacuolar aspartylprotease required for the activation of other vacu-olar proteases, such as carboxypeptidase Y andproteinase B. Proteinase B, prior to processingand activation by proteinase A, has about half theactivity of the processed enzyme. The PRB1 genecodes for proteinase B. Therefore, pep4 mutantsdisplay a substantial decrease or elimination inproteinase A and carboxypeptidase Y activities,and partial reduction in proteinase B activity. Inthe prb1 mutant, only proteinase B activity iseliminated, whereas pep4 prb1 double mutantsshow a substantial reduction or elimination in allthree of these protease activities.

6. Expression VectorsPlasmid vectors designed for heterologous pro-

tein expression in P. pastoris have several com-mon features (Table 2). The foreign geneexpression cassette is one of those and is composedof DNA sequences containing the P. pastorisAOX1 promoter, followed by one or more uniquerestriction sites for insertion of the foreign gene,followed by the transcriptional terminationsequence from the P. pastoris AOX1 gene thatdirects efficient 3' processing and polyadenylationof the mRNAs. Many of these vectors also includethe P. pastoris HIS4 gene as a selectable marker

Table 1P. pastoris Expression Host Strains

StrainName Genotype Phenotype ReferenceY-11430 Wild type NRRL*GS115 his4 Mut+ His– 15KM71 aox1∆::SARG4 his4 arg4 Muts His– 16MC100-3 aox1∆::SARG4 aox2∆::Phis4

his4 arg4 Mut– His– 18SMD1168 pep4∆ his4 Mut+ His–, protease-deficient 10,11SMD1165 prb1 his4 Mut+ His–, protease-deficient 10,11SMD1163 pep4 prb1 his4 Mut+ His–, protease-deficient 10,11

*Northern Regional Research Laboratories, Peoria, IL.



Table 2Common P. pastoris Expression Vectors

Vector Selectablename Targeting markers Features References

pHIL-D2 Intracellular HIS4 NotI sites for AOX1 gene Sreekrishna, replacment personal comm.

pAO815 Intracellular HIS4 Expression cassette sites 2 bounded by BamHI and BglII for generation of multicopy expression vector

pPIC3K Intracellular HIS4 and kanr Multiple cloning sites; for insertion of foreign genes; G418 selection for multicopy strains

pPICZ Intracellular bler Multiple cloning sites for insertion of foreign genes; 10 Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags

pHWO10 Intracellular HIS4 Expression controlled 29 by constitutive GAPp

pGAPZ Intracellular bler Expression controlled Invitrogen, by constitutive GAPp; multiple Carlsbad, CA cloning site for insertion of foreign genes; Zeocin selection for multicopy strains; potential for fusion of foreign protein to His6 and myc epitope tags

pHIL-S1 Secreted HIS4 AOX1p fused to PHO1 Sreekrishna, secretion signal; XhoI, EcoRI, personal comm; and BamHI sites available for Invitrogen, insertion of foreign genes Carlsbad, CA

pPIC9K Secreted HIS4 and kanr AOX1p fused to α-MF prepro 32 signal sequence; XhoI (not unique), EcoRI, NotI, SnaBI, and AvrII sites available for insertion of foreign genes; G418 selection for multicopy strains

pPICZα Secreted bler AOX1p fused to α-MF prepro 10 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

pGAPZα Secreted bler Expression controlled by Invitrogen, constitutive GAPp; GAPp Carlsbad, CA fused to α-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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for transformation into his4 mutant hosts of P.pastoris, as well as sequences required for plasmidreplication and maintenance in bacteria (i.e., ColE1replication origin and ampicillin-resistance gene).Some vectors also contain AOX1 3' flankingsequences that are derived from a region of the P.pastoris genome that lies immediately 3' of theAOX1 gene and can be used to direct fragmentscontaining a foreign gene expression cassette tointegration at the AOX1 locus by gene replacementor gene insertion 3' to AOX1 gene (10,11).

Additional features that are present in certainP. pastoris expression 2vectors serve as tools forspecialized functions. For secretion of foreignproteins, vectors have been constructed that con-tain a DNA sequence immediately following theAOX1 promoter that encodes a secretion signal.The most frequently used of these is the S.cerevisiae α-factor prepro signal sequence(10,11,27,28). However, vectors containing thesignal sequence derived from the P. pastoris acidphosphatase gene (PHO1) are also available.

Vectors with dominant drug-resistance mark-ers that allow for enrichment of strains thatreceive multiple copies of foreign gene expres-sion cassettes during transformations have beendeveloped. One set of vectors (pPIC3K andpPIC9K) contains the bacterial kanamycin-resis-tance gene and confers resistance to high levelsof G418 upon strains that contain multiple cop-ies of these vectors (10,11,28). Another set ofvectors (the pPICZ series) contains the Sh blegene from Streptoalloteichus hindustanus(10,11). This gene is small (375 bp) and confersresistance to the drug Zeocin in E. coli, yeasts(including P. pastoris), and other eukaryotes.Because the ble gene serves as the selectablemarker for both E. coli and P. pastoris, theZeoR vectors are much smaller (approx 3 kb)and easier to manipulate than other P. pastorisexpression vectors. These vectors also containa multiple cloning site (MCS) with severalunique restriction sites for convenience of for-eign gene insertion and sequences encoding theHis6 and myc epitopes so that foreign proteinscan be easily epitope-tagged at their carboxyltermini, if desired.

Another feature present on certain vectors (e.g.,pAO815 and the pPICZ vector series) is designedto facilitate the construction of expression vectorswith multiple expression cassette copies (10).Multiple copies of an expression cassette areintroduced in these vectors by inserting an expres-sion cassette bounded by a BamHI and a BglII siteinto the BamHI site of a vector already containinga single expression cassette copy. The resultingBamHI/BglII junction between the two cassettescan no longer be cleaved by either enzyme allow-ing for the insertion of another BamHI–BglII-bounded cassette into the same vector to generatea vector with three cassette copies. The process ofaddition is repeated until 6–8 copies of a cassetteare present in a single final vector, which is thentransformed into the P. pastoris host strain.

Recently, vectors containing alternative pro-moters have become available (Table 2). Unlikethe AOX1 promoter, these promoters do notrequire induction by methanol, which may beproblematic in some instances. One is a strongconstitutive promoter derived from the P. pastorisglyceraldehyde-3-phosphate dehydrogenase gene(GAP) (29). In addition to not involving metha-nol, the GAP promoter is convenient since cul-tures do not need to be shifted from one carbonsource to another to induce expression of a for-eign gene. However, this promoter is not suitablefor the expression foreign genes whose productsare toxic to P. pastoris since the constant high-level expression can result in the selection ofnonexpressing derivative strains that have eitherlost the expression vector or have gained muta-tions that reduce or eliminate expression of theforeign gene.

The second promoter is from the P. pastorisformaldehyde dehydrogenase gene (FLD1) (30).FLD1 is required for growth of P. pastoris on ei-ther methanol as a sole carbon source or certainmethylated amines such as methylamine as a solenitrogen source and is strongly induced by eitherof these conditions. As a result, expression of aforeign gene placed under control of the FLD1promoter is repressed in media containing glucoseand ammonium ions but can be independentlyinduced using media containing either methanol



or methylamine. With the FLD1 promoter, onlyone expression vector and strain need be con-structed to examine expression under both theseconditions. Importantly, both the GAP and FLD1promoters express foreign genes at levels compa-rable to those observed with the AOX1 promoter.

7. Integration of Vectors into the Pichia pastoris Genome

As in S. cerevisiae, linear vector DNAs cangenerate stable transformants of P. pastoris viahomologous recombination between sequencesshared by the vector and host genome(10,11,15,25). Such integrants show strong stabil-ity in the absence of selective pressure even whenpresent as multiple copies. All P. pastoris expres-sion vectors carry at least one P. pastoris DNAsegment (the promoter fragment) with uniquerestriction sites that can be cleaved and used todirect the vector to integrate into the host genomeby a single crossover type insertion event. Vec-tors containing the P. pastoris HIS4 gene can alsobe directed to integrate into the P. pastorisgenomic his4 locus.

Expression vectors that contain 3' AOX1sequences can be integrated into the P. pastorisgenome by a single crossover event at eitherAOX1 or HIS4 loci or by a gene replacement (Ωinsertion) event at AOX1. The latter event arisesfrom crossovers at both the AOX1 promoter and3' AOX1 regions of the vector and genome, andresults in the deletion of the AOX1 coding region(i.e., gene replacement). Transformants resultingfrom such an AOX1 replacement event are pheno-typically His+ and Muts. Muts strains sometimesexpress higher levels of foreign protein (10). Inaddition, a Muts phenotype serves as a convenientindicator to confirm the presence of an integratedexpression cassette in the P. pastoris genome.

With either single crossover or gene replace-ment integration strategies and selection for His+

transformants, a significant percentage oftransformants will not contain the expression vec-tor. This appears to be due to gene conversionevents between the HIS4 gene on the vector andthe P. pastoris his4 locus such that the wild-typeHIS4 gene recombines into the genome without

any additional vector sequences. These eventsaccount for 10–50% of His+ transformant colo-nies and appear to occur at highest frequencywhen using electroporation to introduce vectorDNAs.

Multiple gene insertion events at a single locusoccur spontaneously at a low but detectable fre-quency—between 1 and 10% of His+ transfor-mants (10,11,31,32). Multicopy events can occuras gene insertions either at the AOX1 or his4 lociand can be detected by DNA analysis methods(e.g., PCR, Southern/dot blotting, or differentialhybridization) (32–34) or by methods that directlyexamine levels of the foreign protein (e.g., activityassay, SDS-PAGE, or colony immunoblotting)(31,32,35). It is possible to enrich transformantpopulations for ones that have multiple copies ofan expression vector by use of either a G418R

or ZeoR gene-containing vector and selectingfor hyper-resistance to the appropriate drug(10,11,28,32). It is important to note that, with theG418R vectors, it is essential to first select forHis+ transformants and to then screen for ones thatare resistant to G418R (32). With ZeoR vectors, itis possible to directly select for hyper-Zeo-resis-tant transformants (10). Drug-resistant strains re-sulting from either the G418R or ZeoR selectionmethods can contain between one and five copiesof the expression vector. To find strains with 20or more copies, it is usually necessary to screenmore than 100 drug-resistant strains.

8. Posttranslational ModificationsP. pastoris has the potential to perform many

of the posttranslational modifications typicallyassociated with higher eukaryotes. These includeprocessing of signal sequences (both pre- andprepro-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 fungican be problematic. In mammals, O-linked oli-gosaccharides are composed of a variety of sug-ars including N-acetylgalactosamine, galactose,and sialic acid. In contrast, lower eukaryotes,including P. pastoris, add O-oligosaccharidessolely composed of mannose (Man) residues

30 Cregg et al.


(4,10,11). The number of Man residues per chain,their manner of linkage, and the frequency andspecificity of O-glycosylation in P. pastoris haveyet to be determined. One should not assume that,because a protein is not O-glycosylated by itsnative host, P. pastoris will not glycosylate it. P.pastoris added O-linked mannose to approx 15%of human IGF-1 protein, although this protein isnot glycosylated at all in humans. Furthermore,one should not assume that the specific Ser andThr 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 isalso different than in higher eukaryotes (4,10,11). Inall eukaryotes, it begins in the endoplasmic reticulumwith the transfer of a lipid-linked oligosaccharide unit(Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = N-acetylglucosamine) to asparagine at the recognitionsequence Asn-X-Ser/Thr. This oligosaccharide coreunit is subsequently trimmed to Man8GlcNAc2. It isat this point that lower and higher eukaryoticglycosylation patterns begin to differ. The mam-malian Golgi apparatus performs a series of trim-ming and addition reactions that generatesoligosaccharides composed of either Man5–6GlcNAc2 (high-mannose type), a mixture of sev-eral different sugars (complex type), or acombination of both (hybrid type). Two distinctpatterns of N-glycosylation have been observedon foreign proteins secreted by P. pastoris. Someproteins, such as S. cerevisiae invertase, are se-creted with carbohydrate structures similar in sizeand structure to the core unit (Man8–11GlcNAc2)(26,36).

Other foreign proteins secreted from P.pastoris receive much more carbohydrate and ap-pear by SDS-PAGE and Western blotting to behyperglycosylated (10,11,23). Interestingly, P.pastoris does not appear to be capable of addingα1,3-terminal mannose to oligosaccharides (R.Trimble, personal communication). This contrastswith S. cerevisiae oligosaccharides where α1,3-linked terminal mannose is common. Aside fromthe probable absence of α1,3-linked mannose, littleis known regarding the structure of P. pastorisouter-chain oligosaccharides. Furthermore, it isalso not clear why outer chains are added to some

P. pastoris-secreted proteins and not others, norhow outer chain addition may be prevented.

N-linked high-mannose oligosaccharidesadded to proteins by yeasts represent a significantproblem in the use of foreign-secreted proteins bythe pharmaceutical industry. They can be exceed-ingly antigenic when introduced intravenouslyinto mammals and are rapidly cleared from theblood by the liver. An additional problem causedby the differences between yeast and mammalianN-linked glycosylation patterns is that the longouter chains can potentially interfere with thefolding or function of a foreign protein.

9. Expression in Fermentor CulturesAlthough a few foreign proteins have expressed

well in P. pastoris shake-flask cultures, expressionlevels in shake-flasks are typically low relative towhat is obtainable in fermenter cultures. One rea-son fermenter culturing is necessary is that only inthe controlled environment of a fermenter is it pos-sible to grow the organism to high cell densities(>100 g/L dry cell weight or 500 OD600 units/mL).Especially for secreted proteins, the concentrationof product in the medium is roughly proportionalto the concentration of cells in culture. A secondreason is that the level of transcription initiatedfrom the AOX1 promoter can be 3–5 times greaterin P. pastoris cells fed methanol at growth-limit-ing rates in fermenter culture relative to cells grownin excess methanol. Thus, even for intracellularlyexpressed proteins, yields of product from a givenstrain as a percentage of total cellular proteins aresignificantly higher from fermenter cultured cells.A third reason is that methanol metabolism utilizesoxygen at a high rate, and expression of foreigngenes is negatively affected by oxygen limitation.Only in the controlled environment of a fermenteris it feasible to accurately monitor and adjust oxy-gen levels in the culture medium. Thus, most usersof the P. pastoris expression system should expectto produce their foreign protein in fermenters.

A hallmark of the P. pastoris system is the easeby which expression strains scale up from shake-flask to high-density fermenter cultures. Consid-erable effort has gone into the optimization ofhigh-cell-density fermentation techniques for



Table 3Heterologous Proteins Expressed in Pichia.pastoris

CommentsProtein Mode, amount, signal sequence References

BacteriaBacillus licheniformis α-amylase S, 2.5 g/L, SUC2 53, 54Bacillus stearothermophilus S, 100 mg/L, native 55D-alanine carboxypeptidaseBordetella pertussis pertussis pertactin (P69) I, 3 g/L 34Clostridium botulinum neurotoxin I, 78 mg/L 56 (BoNT) serotype A and BClostridium botulinum neurotoxin I, 390 µg/g 57 heavy chain fragment, serotype BClostridium botulinum neurotoxin I, 2.4 mg total 58 serotype A binding domainClostridium tetani tetanus toxin fragment C I, 12 g/L 33Escherichia coli acid S, 28.9 U/mg 59 phosphatase/phytase (appA2)Escherichia coli β-galactosidase I, 2.0 × 103 U/mg 16Escherichia coli β-lactamase I 29Leishmania major cathepsin B-like protease S, α-MF 60Staphylococcus aureus staphylokinase S, 50 mg/L, α-MF 61Streptococcus equisimili streptokinase I, 77 mg/L 62Streptomyces subtilisin inhibitor S 63Streptomyces viridosporus T7A S, 2.47 g/L total protein, α-MF 64 peroxidase, endoglucanaseToxoplasma gondii SAG1 antigen S, 12 mg/L, α-MF 65Vibrio cholerae accessory S, 7 mg/L, α-MF 66 cholera enterotoxin (Acc)FungiAlternaria Alt 1 allergen S, α-MF 67Aspergillus awamori glucoamylase S, 400 mg/L, native 68Aspergillus awamori glucoamylase S, 400 mg/L, PHO1 69 catalytic domainAspergillus fumigatus catalase L S, 2.3 g/L, PHO1 70Aspergillus fumigatus dipeptidyl S, PHO1 71 peptidase IV (DPP IV)Aspergillus fumigatus dipeptidyl S, 0.15 mg/L, PHO1 72 peptidase V (DPP V)Aspergillus giganteus α-sarcin ribotoxin S, 1 mg/L, synthetic native, PHO1 73Aspergillus niger phytase (phyA) S, 65 U/ml, α-MF 74Candida guilliermondii xylose reductase gene (xylI) I, 0.65 U/mg; S, 0.18 U/mg, α-MF 75Candida rugosa lipase 1 (CRL) S, 150 U/ml, α-MF 76Fusarium solani pectate lyase (pelC) S, 1 mg/L, PHO1 77Fusarium solani pectate lyase (pelD) S, native 78Geotrichum candidum lipases isoenzymes S, 60 mg/L, α-MF 79Phytophthora cryptogea β-cyptogein S, 45 mg/L, PHO1 80Rhizopus oryzae lipase S, 60 mg/L, α-MF 81Saccharomyces cerevisiae invertase S, 2.5 g/L, native 23Saccharomyces cerevisiae Ktr1p S, 400 mg/L, PHO1 82Saccharomyces cerevisiae S, 40 mg/L, PHO1 82

32 Cregg et al.


(α-1,2-mannosyltransferase)Schizophyllum commune vitamin B2- S, 120 mg/L, α-MF 83 aldehyde-forming enzymeTrametes versicolor (white rot fungus) S, native and α-MF 84 laccase (lccI)Trichoderma harzianum β-(1-6)-glucanase S, 9.3 mg/L 85

ProtistsChondrus crispus red alga hexose oxidase I 86Gracilariopsis lemaneiformis red alga I 87

α-1,4-glucan lyase (GLq1)Plasmodium falciparum merozoite S, 24 mg/L, α-MF 40 surface protein 1 (MSP-1)Plasmodium vivax apical S, 50 mg/L, PHO1 88 Membrane antigen I (AMA-1)Reticulomyxa filosa (giant freshwater amoeba) I, 400 µg/g 89

α2, β2 tubulin isoformsTrypanosoma cruzi acid α-mannosidase S, 11.5 µg/L, native 90

PlantsAllium sativum (garlic) alliin lyase I, 2.167 U/g 91Arabidopsis thaliana I, 18 µg/g 92, 93 NADH:nitrate reductaseBarley (Hordeum vulgare) sucrose S, α-MF 94 fructan 6-fructosyl transferaseBarley α-amylase 1 S, 50 mg/L, native 95Barley α-amylase 2 S, 1 mg/L, native 95Barley aleurone tissue α-glucosidase S, α-MF 96Coffee bean α-galactosidase S, 400 mg/L, α-MF 97Cynara cardunculus (cardoon) cyprosin S, 1 mg/L, native 98Cynodon dactylon (Bermuda grass) Cyn d 1 S, 1.5 g/L, PHO1 99, 100Galanthus nivalis agglutinin S, PHA-E 101Hevea brasiliensis hydroxynitrile lyase I, 22 g/L 102Hevea brasiliensis Hev b 7 patatin-like allergen S, 10 mg/L, α-MF 103, 104Maize cytokinin oxidase S, native 105Oat phytochrome A, phA I, 30 µg/g 106, 107Oat phytochrome A, phyA65 apoprotein I, 20 µg/g 108Olea europaea (olive tree) aeroallergen Ole e 1 S, 60 mg/L, α-MF 109Pepper endo-β-1,4-glucanase cCel1 S, α-MF 110Pepper endo-β-1,4-glucanase cCel2 S, native 110Persea americana (avocado) S, 50 mg/L, α-MF 111 prs a 1 major allergenPhaseolus vulgaris agglutinin S, native 101 (phytohaemagglutinin)Phleum pratense β-expansin S, α-MF 112Potato phytochrome B I, 25 µg/g 113Ragweed allergen Amb a 6 S, 1 mg/L, α-MF 114Soybean root nodule acid phosphatase S, 10 mg/L, α-MF 115Spinach glycolate oxidase I, 250 U/g 116, 117Spinach phosphoribulokinase I, 0.5 mg/g 118Sugar beet defensin AX2 S, PHO1 119Timothy grass group I allergen S, α-MF 120Tomato Lycopesicon esculentum Mill. S, PHO1 121 LeMir (L. esculentum miraculin)Wheat lipid transfer protein S, 720 mg/L, PHO1 122



InvertebratesAchacina fulica Ferussac S, 0.2 mg/L, native 123 (giant African snail) achacinAplysia californica (marine invertebrate) S, 300 mg/L, α-MF 124 ADP ribosyl cyclaseAqueora victoria (jellyfish) I, S, PHA-E 101,125 green fluorescent proteinBoophilus microplus (cattle tick) Bm86 I, S*, 1.5 g/L, SUC2 126–129Cockroach allergen, Bla g 4 S, 50 mg/L 130Drosophila melanogaster S, 160 mg/L, α-MF 131 angiotensin I-converting enzymeDrosophila melanogaster I (mitochondria) 132 caritine palmitoyltransferaseFirefly luciferase I (peroxisome) 133GAVAC™ vaccine against cattle tick S, 2.0 g/L 134Haementeria ghilanii S, 10 mg/L, α-MF 135 (South American leech) ghilantenHirudo medicinalis (leech) hirudin S, 1.5 g/L, α-MF 136Honeybee odorant-binding protein (ASP2) S, 150 mg/L, native 137Honeybee olfactory protein S, 0.2 g/L, native 138Nippostrongylus brasiliensis 139 (parasitic nematode) nonneuronal secreted S, 27 mg/L, α-MF acetylcholine steraseSpider dragline silk protein I, 663 mg/L 140Tick anticoagulant peptide S, 1.7 g/L 27Yellowjacket venom allergen, Ag5 S, 100 mg/L, α-MF 141

Vertebrates (nonhuman)Bovine enterokinase catalytic domain S, 6.3 mg/L, α-MF 142Bovine follicle-stimulating hormone β-subunit S, 4 µg/ml, α-MF 143Bovine IFN-ω 1 S, 4 mg/L, SUC2 144Bovine lysozyme c2 S, 550 mg/L, native 145Bovine opsin S*, 0.3 mg/L, PHO1 146Bovine pancreatic trypsin inhibitor (aprotinin) S, 930 mg/L, α-MF 147Bovine β-casein I, 1 g/L 148Bovine β-lactoglobulin S, >1 g/L, α-MF 42–44Bovine tissue-type plasminogen activator (tPA) S, 1.1 mg/L, α-MF 149Bovine transcobalamin S, α-MF 150Brushtail possum TNFα S, α-MF 151Bungarus fasciatus (snake) venom S, 2 mg/L, native 152 gland acetylcholinesteraseChicken liver α-N-acetylgalactosaminidase S, 11.6 mg/L, α-MF, PHO1 153Electrophorus electricus S, native 154 acetylcholinesterase AChE type THen lysozyme S, 20 mg/L, α-MF 45Mammalian lipocalin allergen Bos d2 S, mg amounts, native 155Mouse 5HT5A 5-tryptamine receptor S*, 40 pmol/mg, α-MF 156Mouse epidermal growth factor S, 450 mg/L, α-MF 28Mouse gelatin S, 14.8 g/l, α-MF 157Mouse gelatinase B S, 10 mg/L, α-MF 158Mouse lysosomal acid α-mannosidase S, native 159Mouse major urinary protein complex (MUP) S, 270 mg/L, native 160Mouse Mdr3 P-glycoprotein I (membrane-bound), 6 µg/mg 161–163Mouse single-chain Fv fragments (sFv) S, 250 mg/L, a-MF, PHO1 164

34 Cregg et al.


Murine endostatin S, 200 mg/L, α-MF 165Murine Golgi mannosidase IA S, PHO1 166Murine macrophage S, 40 mg/L, α-MF 167 inflammatory protein-2 (MIP-2)Ovine follicle-stimulating hormone (oFSH) S, 22 mg/L, α-MF 168Porcine carboxypeptidase B S, 200 mg/L, α-MF 169Porcine follicle-stimulating hormone S, 10 mg/L, PHO1 170Porcine inhibitor of carbonic S, 5 mg/L, α-MF 171 anhydrase (transferrin family)Porcine leukocyte 12-lipoxygenase I 172Rabbit intestinal peptide transporter (PEPT1) I 173Rabbit intestinal peptide transporter (PEPT2) I 174Rabbit monoclonal single-chain Fv specific for S, 100 mg/L, α-MF 175 recombinant human leukemia inhibitory factorRabbit plasma cholesteryl ester transfer protein S, PHO1 176Rabbit testicular angiotensin-converting enzyme S, PHO1, native 177Rat acetycholinesterase S, 1 mg/L, native 152Rat brain acetylcholinesterase T subunit S, 100 U/L α-MF 178Rat complement regulator, crry S, α-MF 179Rat Golgi sialoglycoprotein MG160 S, 10 mg/L, α-MF 180Rat high-mobility group 1 (HMG 1) S, 50 mg/L, α-MF 181Rat liver mitochondrial carnitine palmitoyl I (mitochondria) 182,183 transferases I and II (CPTI and II)Rat neural cell adhesion molecule S, α-MF 184Rat NO synthase reductase domain I, 25 mg/L 185Rat peroxisomal multifunctional I 186 enzyme (perMFE-II)Rat procathepsin B S, 100 mg/L, α-MF 187,188Sea raven type II antifreeze protein (SRAFP) S, 30 mg/L, α-MF 37,189Shark 17α-hydroxylase/C17,20-lyase I 190Syrian golden hamster prion protein PrPc I, <0.1 mg/L 191

Humansα(1,3/4) Fucosyltransferase S, 30 mg/L, α-MF 126α-1,2-Mannosidase 1B w/o TM domain S, α-MF 192α-N-Acetylgalactosaminidase (α-NAGAL) S, 11.6 mg/L, α-MF 193α1-Antitrypsin (α1-AT) S, inulinase signal sequence 194α2-Antiplasmin I 195β2-Adrenergic receptor S*, 25 nmol/g, α-MF 156µ-Opioid receptor S*, a-MF 196ADAR1, ADAR2, ds-RNA-specific I, 1 mg/L 197 adenosine deaminasesAlzheimer’s disease amyloid precursor protein S, PHO1 198

α, β, and γ-secretase productsAlzheimer’s disease amyloid S, 24 mg/L, 0.1 mg/L, α-MF 199 precursor protein, 2 domainsAmyloid precursor-like protein 2 (APLP2) S, 40 mg/L, α-MF 200Amyloid precursor protein (APP) S, 24 mg/L, PHO1 201–203Amyloid precursor proteins, rAPP695, rAPP770 S, 4.5 + 1 mg/L, native 204Bile salt-stimulated lipase S, 300 mg/L, native, INV 205Bivalent diabody against carcinoembryonic S, 1 mg/L, α-MF 206 antigen (CEA), T-cell coreceptor CD2c-Kit receptor kinase domain I, 0.2 mg/L 207,208Carcinoembryonic antigen S, 20 mg/L, α-MF 209



Caspase-3 I, 1 µg/g 210Cathepsin K S, 38 mg/L, α-MF 211,212Cathepsin L propeptide S, 10 mg/L, α-MF 213,214Cathepsin V S, α-MF 215Cathepsin X S, 5 mg/L, α-MF 216CD38 S, 455 mg/L, α-MF 217CD40 ligand soluble form S, 255 mg/L 218Chimeric B7-2 antibody fusion protein S, 15 mg/L, α-MF 219Chorionic gonadotropin S, 24 mg/L (α), 3mg/L (β), 220

α subunit, β subunit, and αβ heterodimer 16 mg/L (αβ), α-MFCromer blood group antigen S, α-MF 221 decay-accelerating factorCytomegalovirus ppUL44 antigen I, 0.1 mg/mL 222Decay-accelerating factor DAF S, 6 mg/L, α-MF 223 (CD55)-Echovirus-7-receptorDouble-stranded RNA-specific editase I (hREDI) I, 1 mg/L 224Endostatin S, 20 mg/L, α-MF 165,225Erythropoietin (EPO) receptor S, 200 mg/L, PHO1 226Fas ligand S, 100 mg/L, α-MF 227Fibrinogen, 143–411, 143–427 S, 100 mg/L, 75 mg/L, α-MF 228Fibroblast collagenase (proMMP-1) S, 2.3 mg/L, α-MF 229Fibrinogen-420 αEC domain S, α-MF 230Gastric cathepsin E S, 0.6 mg/L, native 231Gelatinase B S, 50 mg/L, α-MF 232Granzyme B S, 1 mg/L, α-MF 233Heart muscle carnitine palmitoyl- I (mitochondria) 234 transferase I (M-CPTI)Insulin S, synthetic signal 235Insulin-like growth factor-1 (IGF-1) S, 600 mg/L, α-MF 236Interferon-γ receptor cytoplasmic domain I 23715N-Interferon τ S, 8–10 mg/L, PHO1 238Interleukin-17 (hIL-17) S, 0.35 mg/L, α-MF 239Intracellular proteinase inhibitor (PI-6) I, 50 mg/L 240Kunitz-type protease inhibitor domain S, 1.0 g/L, α-MF of protease nexin-2/amyloid β-protein precursor 241Leukemia inhibitory factor (LIF) S, 17 mg/L, α-MF 242Lymphocyte surface antigen CD38 S, 400 mg/L, PHO1 243Lysosomal α-mannosidase S, 83 µg/L, native 244Mast cell tryptase S, 6.5 mg/L, α-MF 245,246MHC class II heterodimers S, 400 µg/L, α-MF (soluble form/HLA-DR2) 247Monoclonal single-chain Fv S, 50 mg/L, α-MF 248Monocyte chemoattractant protein-1 (MCP-1) S, 100 mg/L, native or α-MF 249Monocyte chemotactic protein-3 (hMCP-3) S, 1 mg/L, PHO1 250NEFA (DNA binding, EF-hand, S, 100–1200 mg/L, α-MF 251

Acidic amino acid rich region)Neural cell adhesion molecule (NCAM) S, 50 mg/L, PHO1 252NonO nucleic acid binding protein I (endoplasmic reticulum) 253Oncostatin-M S, 50 mg/L, α-MF 254Pancreatic α-amylase S, 20 mg/L, α-MF 255Pancreatic triglyceride lipase S, 75 ml/L, PHO1 256Papain nitrile hydratase S, 5 mg/L, α-MF 257Placental alkaline phosphatase (PLAP) S, 2 mg/L, PHO1 258

36 Cregg et al.


Placental protein-14 (PP-14) S, α-MF 259Plasminogen kringles 1-4 S, 17 mg/L, PHO1 260Plasminogen kringles 1-4, angiostatin protein S, 10% total protein, PHO1 261Plasminogen kringles K2 S, 160 mg/L, α-MF 262Procarboxypeptidase A2 S, 180 mg/L, α-MF 263Procathepsin B S, 20 mg/L, α-MF 264Procolipase S, 30 mg/L, native 265Protein kinase C interacting protein-1 (PKCI-1) I, 0.25 mg/L 266Proteinase 3, Wegener’s antigen S, 670 mg/L, α-MF 267Proteinase inhibitor 8 I, 15% total protein 268scFv (against ovarian carcinoma)- S 269,270 biotin mimetic peptidescFv (against squamous carcinoma) S, 50 mg/L, α-MF 248Serum albumin S, 3 g/L, native 24,271–274Serum transferrin N-Lobe S, 240 mg/L, α-MF 275–277Sex steroid binding protein S, 4 mg/L, α-MF 278Single-chain urokinase-type S, 5 mg/L, pre Mucor pusillus plasminogen activator rennin signal 279Thrombomodulin S 280Tissue factor extracellular domain S, 10 mg/L, PHO1 281Tissue kallikrein S, 30 mg/L, α-MF 282,283Tissue-type plasminogen activator S, 170 mg/L, α-MF 18,48 kringle 2 domain 284–287Transforming growth factor S, 10 mg/L, α-MF 288

β receptor extracellular domainTumor necrosis factor α (TNF) I, 10 g/L 31,289Type 1 plasminogen activator inhibitor (PAI-1) S, 3 mg/L, α-MF 290Type III collagen (with prolyl 4-hydroxylase) I, 15 mg/L 291Urokinase-type plasminogen activator-annexin V S, 600 IU/mL, pre Mucor 292

chimera pussils renninVascular endothelial growth factor (VEGF165) S, 40 mg/L, PHO1 293

VirusesA/VICTORIA/3/75 influenza virus S, 3 mg/ml, α-MF 294, 295 neuraminidase head domainBovine herpes virus-1 glycoprotein D S, 20 mg/L, α-MF 296, 297Dengue virus type 1 structural S, PHO1, prM virus signal sequence 298 gene recombinant E proteinHepatitis B virus surface antigen I, 400 mg/L 25, 299Hepatitis B virus surface antigen-HIV gp41 epitope chimera I 300Hepatitis E virus ORF3 I 301Human immunodeficiency gp120 (ENV) S, 20 mg/L, α-MF 302 virus type 1 (HIV-1) envelope glycoprotein,Polyomavirus large T antigen I, 0.5 mg/L 303Reovirus λ 1 core protein I, 0.8 mg/L 304Reovirus σ 1 protein I 305Vaccinia virus complement control protein S, 3 mg/L, a-MF 41

I = Intracellular (with subcellular location), S = Secreted, S* = Secreted to plasma membrane. Amounts are highest reported forparticular protein. Signal sequences: α-MF (S. cerevisiae α-mating factor); PHO1 (P. pastoris acid phosphatase); SUC2(S. cerevisiae invertase).



expression strains, and, as a result, a variety offed-batch and continuous culture schemes areavailable (10,11). All schemes involve the initialgrowth of strains in a defined medium on glyc-erol. During this period, growth is rapid but het-erologous gene expression is fully repressed.Upon depletion of glycerol, a transition phase isinitiated in which additional glycerol is fed to cul-tures at a growth-limiting rate. Finally, methanol,or a mixture of glycerol and methanol, is fed tocultures to induce expression. The time of harvest,typically the peak concentration of a foreign pro-tein, is determined empirically for each protein.

High-density fermentation of P. pastoris ex-pression strains is especially attractive for the pro-duction of secreted proteins, because theirconcentration in the culture medium should in-crease with cell density. Unfortunately, the con-centrations of other cellular materials, particularlyproteases, increase as well. Three strategies haveproven effective in minimizing the proteolytic in-stability of foreign proteins secreted into the P.pastoris culture medium. One is the addition ofamino acid-rich supplements, such as peptone orcasamino acids, to the culture medium which ap-pear to reduce product degradation by acting asexcess substrates for one or more problem pro-teases (28). A second is changing the culture me-dium pH (28). P. pastoris is capable of growingacross a relatively broad pH range from 3.0 to 7.0which allows considerable leeway in adjusting thepH to one that is not optimal for a problem pro-tease. A third is the use of a protease-deficient P.pastoris host strain (10,11).

10. Expression of Proteinfor Structural Studies

The P. pastoris yeast expression system hasgained widespread use for production of proteinsfor structural studies. Proteins that are normallysecreted or require mammalian post-translationalmodifications can not be produced in an activeform in E. coli. For these proteins in particular,large quantities of active protein can be producedin P. pastoris. Since P. pastoris grows rapidly oninexpensive media similar to E. coli, it is feasibleto quickly and cheaply synthesize and examine

large numbers of mutant proteins suitable forstructure/function studies. The P. pastoris yeastexpression system offers key advantages relativeto other mammalian expression systems and toBacculovirus. In particular, P. pastoris grows inminimal medium that can be modified for produc-tion of 15N-labeled proteins (37-41). Even triplylabeled (15N, 13C, 2H) proteins have been pro-duced in this way (42,43). In some cases, theyields of labeled proteins compete favorably withthose obtained from E. coli (44–46). Another ad-vantage of the P. pastoris yeast expression sys-tem is that glycosylated proteins can be preparedin high yields and the sugars that are attached byP. pastoris are of a simple high mannose form(47,48). These sugars are easily removed bycleavage with endoglycosidase H, leaving thecore GlcNac if advantage for X-ray crystallo-graphic analysis where a homogeneous proteinsample is required (49–50). Indeed, crystallogra-phy of glycosylated proteins can be extremely dif-ficult if the protein is produced in mammaliancells or in Bacculovirus because of incomplete re-moval of the complex sugars that these organismsattach. A comprehensive list of heterologous pro-teins expressed in P. pastoris can be found inTable 3 and at www.kgi.edu/html/noncore/pro-gram 4.htm#jc.

11. Pichia methanolica:New Kid on the Block

Recently P. methanolica, another methylo-trophic species, has been developed as a heterolo-gous expression system (51,52). The essential fea-tures of an expression system have been developedfor this yeast, including, strains, expression vec-tors, transformation system (mostly via non-ho-mologous recombination), a methanol-regulatedpromoter for recombinant protein expression, andfermentation techniques. A possible advantageover P. pastoris is in fermentation; biomass gen-eration during fermentation can use glucose insteadof glycerol and less methanol is required for theinduction of recombinant protein expression. Themain disadvantage of P. methanolica is that itsrelatively new arrival as an expression tool meansthat it is less well characterized, including opti-

38 Cregg et al.


mization of parameters for transformation, pro-tein expression, and fermentation. Similar to thesituation with P. pastoris in which its use is cov-ered by patents that are owned by RCT, patentscovering the use of P. methanolica are owned byZymoGenetics, Inc. of Seattle, WA. Althoughuse for academic research purposes is permitted,a license is required for commercial use after aninitial one-year commercial evaluation period.Also like P. pastoris, P. methanolica has been li-censed to Invitrogen Corp., Carlsbad, CA for dis-tribution to academic and commercial users. P.methanolica strains, plasmids, and licensing in-formation can be obtained from Invitrogen.

12. SummaryBased on available data, there is an approx 50–

75% probability of expressing any protein of in-terest in P. pastoris at a reasonable level. Thebiggest hurdle seems to be generating initial suc-cess—that is, expressing a specific protein at anylevel. After success at this stage, there are well-defined parameters that can be manipulated tooptimize expression, and it is often at this stagethat attractive levels of expression are achieved.

Although there are relatively few examples ofexpression of ≥10 g/L, there are many examplesof expression in the ≥1 g/L range, ranking the P.pastoris expression system as one of the most pro-ductive eukaryotic expression systems available.There are also examples of proteins that have beensuccessfully expressed in P. pastoris that werecompletely unsuccessful in Baculovirus or S.cerevisiae expression systems, making the P.pastoris system an important alternative to haveavailable in the protein expression "toolbox." SeeTable 3 for a comprehensive list of heterologousproteins expressed in P. pastoris.

AcknowledgmentsThe preparation of this manuscript was supported

by Grant DK-43698 from the National Institutes ofHealth and Grant ER20334 from the Department ofEnergy, Office of Basic Energy Sciences (toJ.M.C.). We would like to thank Terrie Hadfield,Oregon Graduate Institute, for expert assistance inpreparation of the manuscript, and Elizabeth

Komives, University of California, San Diego, forinformation on heavy atom labeled protein.

References1. Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992)

Foreign gene expression in yeast: a review. Yeast 8,423–488.

2. Cregg, J. M., Vedvick, T. S., and Raschke, W. C.(1993) Recent advances in the expression of foreigngenes in Pichia pastoris. Biotechnology (NY) 11,905–910.

3. Romanos, M. (1995) Advances in the use of Pichiapastoris for high–level expression. Curr. Opin.Biotechnol. 6, 527–533.

4. Cregg, J. M. (1999) Expression in the methylotrophicyeast Pichia pastoris. In Gene Expression Systems:Using Nature for the Art of Expression (Fernandez, J.M. and Hoeffler, J. P., eds.), Academic Press, San Di-ego, CA, pp. 157–191.

5. Cregg, J. M. and Higgins, D. R. (1995) Production offoreign proteins in the yeast Pichia pastoris. Can. J.Bot. 73(Suppl. 1), S981–S987.

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 se-cretion of heterologous proteins in the methylotrophicyeast Pichia pastoris. Gene 190, 55–62.

7. Gellissen, G. and Hollenberg, C. P. (1997) Applica-tion of yeasts in gene expression studies: a compari-son of Saccharomyces cerevisiae, Hansenulapolymorpha and Kluyveromyces lactis—a review.Gene 190, 87–97.

8. Higgins, D. R. (1995) Overview of protein expres-sion in Pichia pastoris. In Current Protocols in Pro-tein Science, Supplement 2 (Wingfield, P. T., ed.),John Wiley and Sons, New York, pp. 5.7.1–5.7.16.

9. Sreekrishna, K. (1993) Strategies for optimizingprotein expression and secretion in themethylotrophic yeast Pichia pastoris. In IndustrialMicroorganisms: Basic and Applied Molecular Ge-netics (Baltz, R. H., Hegeman, G. D., and Skatrud,P. L., ed.), American Society for Microbiology,Washington, DC, pp. 119–126.

10. Higgins, D. R. and Cregg, J. M. (1998) Pichia Proto-cols, Methods in Molecular Biology, vol. 103,Humana Press, Totowa, NJ.

11. Cereghino, J. L. and Cregg, J. M. (2000) Heterolo-gous protein expression in the methylotrophic yeastPichia pastoris. FEMS Microbiol. Rev., 24, 45-66.

12. Ogata, K., Nishikawa, H., and Ohsugi, M. (1969) Ayeast capable of utilizing methanol. Agric. Biol.Chem. 33, 1519–1520.

13. Wegner, G. H. (1990) Emerging applications of themethylotrophic yeasts. FEMS Microbiol. Rev. 7,279–283.



14. Ellis, S. B., Brust, P. F., Koutz, P. J., Waters, A. F.,Harpold, M. M., and Gingeras, T. R. (1985) Isolationof alcohol oxidase and two other methanol regulat-able genes from the yeast Pichia pastoris. Mol. Cell.Biol. 5, 1111–1121.

15. Cregg, J. M., Barringer, K. J., Hessler, A. Y., andMadden, K. R. (1985) Pichia pastoris as a hostsystem for transformations. Mol. Cell. Biol. 5,3376–3385.

16. Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C.A., and Gingeras, T. R. (1987) Expression of the LacZgene from two methanol-regulated promoters inPichia pastoris. Nucleic Acids Res. 15, 3859–3876.

17. Cregg, J. M. and Madden, K. R. (1987) Developmentof yeast transformation systems and construction ofmethanol-utilization-defective mutants of Pichiapastoris gene disruption. In Biological Research onYeasts, vol. 2 (Stewart, G. G., Russell, I., Klein, R.D., and Hiebsch, R. R., ed.), CRC Press, Boca Raton,FL, pp. 1–18.

18. Cregg, J. M., Madden, K. R., Barringer, K. J., Thill,G. P., and Stillman, C. A. (1989) Functional charac-terization of the two alcohol oxidase genes from theyeast Pichia pastoris. Mol. Cell. Biol. 9, 1316–1323.

19. Koutz, P. J., Davis, G. R., Stillman, C., Barringer, K.,Cregg, J. M., and Thill, G. (1989) Structural compari-son of the Pichia pastoris alcohol oxidase genes.Yeast 5, 167–177.

20. Kurtzman, C. P. and Robnett, C. J. (1998) Identifica-tion and phylogeny of ascomycetous yeasts fromanalysis of nuclear large subunit (26S) ribosomalDNA partial sequences. Antonie van Leeuwenhoek73, 331–371.

21. Veenhuis, M., van Dijken, J. P., and Harder, W.(1983) The significance of peroxisomes in the me-tabolism of one-carbon compounds in yeasts. Adv.Microb. Physiol. 24, 1–82.

22. Couderc, R. and Baratti, J. (1980) Oxidation ofmethanol by the yeast Pichia pastoris: purificationand properties of alcohol oxidase. Agric. Biol. Chem.44, 2279–2289.

23. Tschopp, J. F., Sverlow, G., Kosson, R., Craig, W.,and Grinna, L. (1987) High level secretion ofglycosylated invertase in the methylotrophic yeastPichia pastoris. Biotechnology (NY) 5, 1305–1308.

24. Barr, K. A., Hopkins, S. A., and Sreekrishna, K.(1992) Protocol for efficient secretion of HSA devel-oped from Pichia pastoris. Pharm. Eng. 12, 48–51.

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 as-sembly of hepatitis B surface antigen in themethylotrophic yeast, Pichia pastoris. Biotechnology(NY) 5, 479–485.

26. Chirulova, V., Cregg, J. M., and Meagher, M. M.(1997) Recombinant protein production in an alcoholoxidase-defective strain of Pichia pastoris in fed-batch fermentations. Enzyme Microb. Technol. 21,277–283.

27. Laroche, Y., Storme, V., De Muetter, J., Messens,J., and Lauwereys, M. (1994) High-level secretionand very efficient isotopic labeling of tick antico-agulant peptide (TAP) expressed in themethylotrophic yeast Pichia pastoris. Biotechnology(NY) 12, 1119–1124.

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) Produc-tion of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strainscontaining multiple gene copies. Gene 105, 205–212.

29. Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S.V., and Cregg, J. M. (1997) Isolation of the Pichiapastoris glyceraldehyde-3-phosphate dehydrogenasegene and regulation and use of its promoter. Gene186, 37–44.

30. Shen, S., Sulter, G., Jeffries, T. W., and Cregg, J. M.(1998) A strong nitrogen source-regulated promoterfor controlled expression of foreign genes in the yeastPichia pastoris. Gene 216, 93–102.

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) High-level expression, purification, and characterization ofrecombinant human tumor necrosis factor synthe-sized in the methylotrophic yeast Pichia pastoris.Biochemistry 28, 4117–4125.

32. Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos,M. A., and Sreekrishna, K. (1994) Rapid selection us-ing G418 of high copy number transformants of Pichiapastoris for high-level foreign gene expression. Bio-technology (NY) 12, 181–184.

33. Clare, J. J., Rayment, F. B., Ballantine, S. P.,Sreekrishna, K., and Romanos, M. A. (1991) High-level expression of tetanus toxin fragment C in Pichiapastoris strains containing multiple tandem integra-tions of the gene. Biotechnology (NY) 9, 455–460.

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 pro-duction and immunological properties. Vaccine 9,901–906.

35. Wung, J. L. and Gascoigne, N. R. (1996) Antibodyscreening for secreted proteins expressed in Pichiapastoris. Biotechniques 21, 808, 810, 812.

36. Trimble, R. B., Atkinson, P. H., Tschopp, J. F.,Townsend, R. R., and Maley, F. (1991) Structure ofoligosaccharides on Saccharomyces SUC2 invertase

40 Cregg et al.


secreted by the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 266, 22807–22817.

37. Gronwald, W., Loewen, M. C., Lix, B., Daugulis,A. J., Sonnichsen, F. D., Davies, P. L. and Sykes,B. D. (1998) The solution structure of type II an-tifreeze protein reveals a new member of the lec-tin family. Biochemistry 37, 4712–4721.

38. Kirkitadze, M. D., Krych, M., Uhrin, D., Dryden,D. T., Smith, B. O., Cooper, A., Wang, X.,Hauhart, R., Atkinson, J. P., and Barlow, P. N.(1999) Independently melting modules and highlystructured intermodular junctions within comple-ment receptor type 1. Biochemistry 38, 7019–7031.

39. McAlister, M. S., Brown, M. H., Willis, A. C., Rudd,P. M., Harvey, D. J., Aplin, R., Shotton, D. M., Dwek,R. A., Barclay, A. N., and Driscoll, P. C. (1998)Structural analysis of the CD5 antigen—expression,disulphide bond analysis and physicalcharacterisation of the CD5 scavenger receptor super-family domain 1. Eur. J. Biochem. 257, 131–141.

40. Morgan, W. D., Birdsall, B., Frenkiel, T. A.,Gradwell, M. G., Burghaus, P. A., Syed, S. E.,Uthaipibull, C., Holder, A. A., and Feeney, J. (1999)Solution structure of an EGF module pair from thePlasmodium falciparum merozoite surface protein 1.J. Mol. Biol. 289, 113–122.

41. Wiles, A. P., Shaw, G., Bright, J., Perczel, A.,Campbell, I. D., and Barlow, P. N. (1997) NMR stud-ies of a viral protein that mimics the regulators ofcomplement activation. J. Mol. Biol. 272, 253–265.

42. Denton, H., Smith, M., Husi, H., Uhrin, D., Barlow,P. N., Batt, C. A., and Sawyer, L. (1998) Isotopi-cally labeled bovine beta-lactoglobulin for NMRstudies expressed in Pichia pastoris. Prot. Exp.Purif. 14, 97–103.

43. Uhrinova, S., Uhrin, D., Denton, H., Smith, M., Saw-yer, L., and Barlow, P. N. (1998) Complete assign-ment of 1H, 13C and 15N chemical shifts for bovinebeta-lactoglobulin: secondary structure and topologyof the native state is retained in a partially unfoldedform. J. Biomol. NMR 12, 89–107.

44. Kim, T. R., Goto, Y., Hirota, N., Kuwata, K., Denton,H., Wu, S. Y., Sawyer, L., and Batt, C. A. (1997)High-level expression of bovine beta-lactoglobulin inPichia pastoris and characterization of its physicalproperties. Protein Eng. 10, 1339–1345.

45. Mine, S., Ueda, T., Hashimoto, Y., Tanaka, Y., andImoto, T. (1999) High-level expression of uniformly15N-labeled hen lysozyme in Pichia pastoris andidentification of the site in hen lysozyme where phos-phate ion binds using NMR measurements. FEBSLett. 448, 33–37.

46. Wood, M. J. and Komives, E. A. (1999) Productionof large quantities of isotopically labeled protein inPichia pastoris by fermentation. J. Biomol. NMR 13,149–159.

47. McAlister, M. S., Davis, B., Pfuhl, M., and Driscoll,P. C. (1998) NMR analysis of the N-terminal SRCRdomain of human CD5: engineering of a glycopro-tein for superior characteristics in NMR experiments.Protein Eng. 11, 847–853.

48. Nilsen, S. L., DeFord, M. E., Prorok, M., Chibber, B.A., Bretthauer, R. K., and Castellino, F. J. (1997)High-level secretion in Pichia pastoris and biochemi-cal characterization of the recombinant kringle 2 do-main of tissue-type plasminogen activator.Biotechnol. Appl. Biochem. 25, 63–74.

49. Greenwald, J., Le, V., Corrigan, A., Fischer, W.,Komives, E., Vale, W., and Choe, S. (1998) Charac-terization of the extracellular ligand-binding domainof the type II activin receptor. Biochemistry 37,16711–16718.

50. Greenwald, J., Fischer, W. H., Vale, W. W., andChoe, S. (1999) Three-finger toxin fold for the extra-cellular ligand-binding domain of the type II activinreceptor serine kinase. Nat. Struct. Biol. 6, 18–22.

51. Raymond, C. K., Bukowski, T., Holderman, S. D.,Ching, A. F. T., Vanaja, E., and Stamm, M. R.(1997) Development of the methylotrophic yeast,Pichia methanolica, for the expression of the 65-kilodalton isoform of human glutamate decarboxy-lase. Yeast 14, 11–23.

52. Raymond, C. K. (1999) Recombinant protein expres-sion in Pichia methanolica. In Gene Expression Sys-tems (Fernandez, J. and Hoeffler, J., ed.), AcademicPress, San Diego, CA, pp. 193–209.

53. Montesino, R., Garcia, R., Quintero, O., and Cremata,J.A. (1998) Variation in N-linked oligosaccharidestructures on heterologous proteins secreted by themethylotrophic yeast Pichia pastoris. Protein Expr.Purif. 14, 197–207.

54. Paifer, E., Margolles, E., Cremata, J., Montesino, R.,Herrera, L., and Delgado, J. M. (1994) Efficient ex-pression and secretion of recombinant alpha amylasein Pichia pastoris using two different signal se-quences. Yeast 10, 1415–1419.

55. Despreaux, C. W. and Manning, R. F. (1993) ThedacA gene of Bacillus stearothemophilus coding forD-alanine carboxypeptidase: cloning, structure andexpression in Escherichia coli and Pichia pastoris.Gene 131, 35–41.

56. Smith, L. A. (1998) Development of recombinantvaccines for botulinum neurotoxin. Toxicon 36,1539–1548.

57. Potter, K. J., Bevins, M. A., Vassilieva, E. V.,Chiruvolu, V. R., Smith, T., Smith, L. A., andMeagher, M. M. (1998) Production and purificationof the heavy-chain fragment C of botulinum neuro-toxin, serotype B, expressed in the methylotrophicyeast Pichia pastoris. Protein Expr. Purif. 13, 357–365.

58. Byrne, M. P., Smith, T. J., Montgomery, V. A., andSmith, L. A. (1998) Purification, potency, and effi-



cacy of the botulinum neurotoxin type A binding do-main from Pichia pastoris as a recombinant vaccinecandidate. Infect. Immun. 66, 4817–4822.

59. Rodriguez, E., Han, Y., and Lei, X. G. (1999) Clon-ing, sequencing, and expression of an Escherichiacoli acid phosphatase/phytase gene (appA2) isolatedfrom pig colon. Biochem. Biophys. Res. Commun.257, 117–123.

60. Chan, V. J., Selzer, P. M., McKerrow, J. H., andSakanari, J. A. (1999) Expression and alterationof the S2 subsite of the Leishmania major cathep-sin B-like cysteine protease. Biochem. J. 340,113–117.

61. Miele, R. G., Prorok, M., Costa, V. A., andCastellino, F. J. (1999) Glycosylation of aspar-agine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a proteinwith highly attenuated plasminogen activator ac-tivity. J. Biol. Chem. 274, 7769–7776.

62. Hagenson, M. J., Holden, K. A., Parker, K. A.,Wood, P. J., Cruze, J. A., Fuke, M., Hopkins, T. R.,and Stroman, D. W. (1989) Expression of streptoki-nase in Pichia pastoris yeast. Enzyme Microb.Technol. 11, 650–656.

63. Markaryan, A., Beall, C. J., and Kolattukudy, P. E.(1996) Inhibition of Aspergillus serine proteinase byStreptomyces subtilisin inhibitor and high-level ex-pression of this inhibitor in Pichia pastoris. Biochem.Biophys. Res. Commun. 220, 372–376.

64. Thomas, L. and Crawford, D. L. (1998) Cloning ofclustered Streptomyces viridosporus T7A lignocellu-lose catabolism genes encoding peroxidase andendoglucanase and their extracellular expression inPichia pastoris. Can. J. Microbiol. 44, 364–372.

65. Biemans, R., Gregoire, D., Haumont, M., Bosseloir,A., Garcia, L., Jacquet, A., Dubeaux, C., and Bollen,A. (1998) The conformation of purified Toxoplasmagondii SAG1 antigen, secreted from engineeredPichia pastoris, is adequate for serorecognition andcell proliferation. J. Biotechnol. 66, 137–146.

66. Trucksis, M., Conn, T. L., Fasano, A., and Kaper, J. B.(1997) Production of Vibrio cholerae accessory chol-era enterotoxin (Ace) in the yeast Pichia pastoris. In-fect. Immun. 65, 4984–4988.

67. De Vouge, M. W., Thaker, A. J., Curran, I. H., Zhang,L., Muradia, G., Rode, H., and Vijay, H. M. (1996)Isolation and expression of a cDNA clone encoding anAlternaria alternata Alt a 1 subunit. Int. Arch. AllergyImmunol. 111, 385–395.

68. Fierobe, H.-P., Mirgorodskaya, E., Frandsen, T. P.,Roepstorff, P., and Svensson, B. (1997) Over-expression and characterization of Aspergillusawamori wild-type and mutant glucoamylase secretedby the methylotrophic yeast Pichia pastoris: compari-son with wild-type recombinant glucoamylase pro-duced using Saccharomyces cerevisiae and Aspergillusniger as hosts. Protein Expr. Purif. 9, 159–170.

69. Heimo, H., Palmu, K., and Suominen, I. (1997) Ex-pression in Pichia pastoris and purification of As-pergillus awamori glucoamylase catalytic domain.Protein Expr. Purif. 11, 304.

70. Calera, J. A., Paris, S., Monod, M., Hamilton, A. J.,Debeaupuis, J. P., Diaquin, M., Lopez-Medrano, R.,Leal, F., and Latge, J. P. (1997) Cloning and disrup-tion of the antigenic catalase gene of Aspergillusfumigatus. Infect. Immun. 65, 4718–4724.

71. Beauvais, A., Monod, M., Wyniger, J., Debeaupuis,J. P., Grouzmann, E., Brakch, N., Svab, J.,Hovanessian, A. G., and Latge, J. P. (1997)Dipeptidyl-peptidase IV secreted by Aspergillusfumigatus, a fungus pathogenic to humans. Infect.Immun. 65, 3042–3047.

72. Beauvais, A., Monod, M., Debeaupuis, J. P., Diaquin,M., Kobayashi, H., and Latge, J. P. (1997) Biochemi-cal and antigenic characterization of a newdipeptidyl-peptidase isolated from Aspergillusfumigatus. J. Biol. Chem. 272, 6238–6244.

73. Martínez-Ruiz, A., Martínez del Pozo, A., Lacadena,J., Mancheño, J. M., Oñaderra, M., López-Otin, C.,and Gavilanes, J. G. (1998) Secretion of recombinantpro- and mature fungal alpha-sarcin ribotoxin by themethylotrophic yeast Pichia pastoris: the Lys-Argmotif is required for maturation. Protein Expr. Purif.12, 315–322.

74. Han, Y. and Lei, X. G. (1999) Role of glycosylationin the functional expression of an Aspergillus nigerphytase (phyA) in Pichia pastoris. Arch. Biochem.Biophys. 364, 83–90.

75. Handumrongkul, C., Ma, D. P., and Silva, J. L. (1998)Cloning and expression of Candida guilliermondiixylose reductase gene (xyl1) in Pichia pastoris. Appl.Microbiol. Biotechnol. 49, 399–404.

76. Brocca, S., Schmidt-Dannert, C., Lotti, M.,Alberghina, L., and Schmid, R. D. (1998) Design, to-tal synthesis, and functional overexpression of theCandida rugosa lip1 gene coding for a major indus-trial lipase. Protein Sci. 7, 1415–1422.

77. Guo, W., Gonzalez-Candelas, L., and Kolattukudy,P. E. (1995) Cloning of a novel constitutively ex-pressed pectate lyase gene pelB from Fusariumsolani f. sp. pisi (Nectria haematococca, matingtype VI) and characterization of the gene productexpressed in Pichia pastoris. J. Bacteriol. 177,7070–7077.

78. Guo, W., Gonzalez-Candelas, L., and Kolattukudy,P. E. (1996) Identification of a novel pelD gene ex-pressed uniquely in planta by Fusarium solani f. sp.pisi (Nectria haematococca, mating type VI) andcharacterization of its protein product as an endo-pectate lyase. Arch. Biochem. Biophys. 332, 305–312.

79. Holmquist, M., Tessier, D. C., and Cygler, M. (1997)High-level production of recombinant Geotrichumcandidum lipases in yeast Pichia pastoris. ProteinExpr. Purif. 11, 35–40.

42 Cregg et al.


80. O’Donohue, M. J., Boissy, G., Huet, J. C.,Nespoulous, C., Brunie, S., and Pernollet, J. C. (1996)Overexpression in Pichia pastoris and crystallizationof an elicitor protein secreted by the phytopathogenicfungus, Phytophthora cryptogea. Protein Expr. Purif.8, 254–261.

81. Minning, S., Schmidt-Dannert, C., and Schmid, R. D.(1998) Functional expression of Rhizopus oryzae li-pase in Pichia pastoris: high-level production andsome properties. J. Biotechnol. 66, 147–156.

82. Romero, P. A., Lussier, M., Sdicu, A. M., Bussey,H., and Herscovics, A. (1997) Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae.Comparison of the enzymic properties of soluble re-combinant Ktr1p and Kre2p/Mnt1p produced inPichia pastoris. Biochem. J. 321, 289–295.

83. Chen, H. and McCormick, D. B. (1997) Riboflavin5'-hydroxymethyl oxidation. Molecular cloning, ex-pression, and glycoprotein nature of the 5'-alde-hyde-forming enzyme from Schizophyllum com-mune. J. Biol. Chem. 272, 20077–20081.

84. Jonsson, L. J., Saloheimo, M., and Penttila, M. (1997)Laccase from the white-rot fungus Trametes versi-color: cDNA cloning of lcc1 and expression in Pichiapastoris. Curr. Genet. 32, 425–430.

85. Bom, I. J., Dielbandhoesing, S. K., Harvey, K. N.,Oomes, S. J., Klis, F. M., and Brul, S. (1998) A newtool for studying the molecular architecture of the fun-gal cell wall: one-step purification of recombinanttrichoderma beta-(1–6)-glucanase expressed in Pichiapastoris. Biochim. Biophys. Acta 1425, 419–424.

86. Hansen, O. C. and Stougaard, P. (1997) Hexose oxi-dase from the red alga Chondrus crispus. Purifica-tion, molecular cloning, and expression in Pichiapastoris. J. Biol. Chem. 272, 11581–11587.

87. Bojsen, K., Yu, S., Kragh, K. M., and Marcussen, J.(1999) A group of alpha-1,4-glucan lyases and theirgenes from the red alga Gracilariopsis lemanei-formis: purification, cloning, and heterologous ex-pression. Biochim. Biophys. Acta 1430, 396–402.

88. Kocken, C. H. M., Dubbeld, M. A., Van Der Wel, A.,Pronk, J. T., Waters, A. P., Langermans, J. A. M.,and Thomas, A. W. (1999) High-level expression ofPlasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris: strong immunogenicity inMacaca mulatta immunized with P. vivax AMA-1and adjuvant SBAS2. Infect. Immun. 67, 43–49.

89. Linder, S., Schliwa, M., and Kube-Granderath, E.(1997) Expression of Reticulomyxa filosa tubulins inPichia pastoris: regulation of tubulin pools. FEBSLett. 417, 33–37.

90. Vandersall-Nairn, A. S., Merkle, R. K., O’Brien, K.,Oeltmann, T. N., and Moremen, K. W. (1998) Clon-ing, expression, purification, and characterization ofthe acid alpha-mannosidase from Trypanosoma cruzi.Glycobiology 8, 1183–1194.

91. Weik, R., Francky, A., Striedner, G., Raspor, P.,Bayer, K., and Mattanovich, D. (1998) Recombinantexpression of alliin lyase from garlic (Allium sativum)in bacteria and yeasts. Planta Med. 64, 387–388.

92. Su, W., Mertens, J. A., Kanamaru, K., Campbell, W.H., and Crawford, N. M. (1997) Analysis of wild-typeand mutant plant nitrate reductase expressed in themethylotrophic yeast Pichia pastoris. Plant Physiol.115, 1135–1143.

93. Su, W., Huber, S. C., and Crawford, N. M. (1996)Identification in vitro of a post-translational regula-tory site in the hinge 1 region of Arabidopsis nitratereductase. Plant Cell 8, 519–527.

94. Hochstrasser, U., Luscher, M., De Virgilio, C., Boler,T., and Wiemken, A. (1998) Expression of a func-tional barley sucrose-fructan 6-fructosyltransferase inthe methylotrophic yeast Pichia pastoris. FEBS Lett.440, 356–360.

95. Juge, N., Andersen, J. S., Tull, D., Roepstorff, P., andSvensson, B. (1996) Overexpression, purification,and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeastPichia pastoris. Protein Expr. Purif. 8, 204–214.

96. Tibbot, B. K., Henson, C. A., and Skadsen, R. W.(1998) Expression of enzymatically active, recombi-nant barley alpha-glucosidase in yeast and immuno-logical detection of alpha-glucosidase from seedtissue. Plant Mol. Biol. 38, 379–391.

97. Zhu, A., Monahan, C., Zhang, Z., Hurst, R., Leng, L.,and Goldstein, J. (1995) High-level expression andpurification of coffee bean alpha-galactosidase pro-duced in the yeast Pichia pastoris. Arch. Biochem.Biophys. 324, 65–70.

98. White, P. C., Cordeiro, M. C., Arnold, D., Brodelius,P. E., and Kay, J. (1999) Processing, activity, and in-hibition of recombinant cyprosin, an aspartic protein-ase from cardoon (Cynara cardunculus). J. Biol.Chem. 274, 16685–16693.

99. Chang, Z. N., Peng, H. J., Lee, W. C., Chen, T. S.,Chua, K. Y., Tsai, L. C., Chi, C. W., and Han, S. H.(1999) Sequence polymorphism of the group 1 aller-gen of Bermuda grass pollen. Clin. Exp. Allergy 29,488–496.

100. Smith, P. M., Suphioglu, C., Griffith, I. J., Theriault,K., Knox, R. B., and Singh, M. B. (1996) Cloningand expression in yeast Pichia pastoris of a biologi-cally active form of Cyn d 1, the major allergen ofBermuda grass pollen. J. Allergy Clin. Immunol. 98,331–343.

101. Raemaekers, R. J. M., de Muro, L., Gatehouse, J. A.,and Fordham-Skelton, A. P. (1999) Functional phy-tohaemagglutinin (PHA) and Galanthus nivalis ag-glutinin (GNA) expressed in Pichia pastoris: correctN-terminal processing and secretion of heterologousproteins expressed using the PHA-E signal peptide.Eur. J. Biochem. 265, 394–403.



102. Hasslacher, M., Schall, M., Hayn, M., Bona, R.,Rumbold, K., Luckl, J., Griengl, H., Kohlwein, S. D.,and Schwab, H. (1997) High-level intracellular ex-pression of hydroxynitrile lyase from the tropical rub-ber tree Hevea brasiliensis in microbial hosts. ProteinExpr. Purif. 11, 61–71.

103. Sowka, S., Wagner, S., Krebitz, M., Arija-Mad-Arif,S., Yusof, F., Kinaciyan, T., Brehler, R., Scheiner,O., and Breiteneder, H. (1998) cDNA cloning of the43-kDa latex allergen Hev b 7 with sequence similar-ity to patatins and its expression in the yeast Pichiapastoris. Eur. J. Biochem. 255, 213–219.

104. Breiteneder, H., Sowka, S., Wagner, S., Krebitz, M.,Hafner, C., Kinaciyan, T., Yeang, H. Y., andScheiner, O. (1999) Cloning of the patatin-like latexallergen Hev b 7, its expression in the yeast Pichiapastoris and its immunological characterization. Int.Arch. Allergy Immunol. 118, 309–310.

105. Morris, R. O., Bilyeu, K. D., Laskey, J. G., andCheikh, N. N. (1999) Isolation of a gene encoding aglycosylated cytokinin oxidase from maize.Biochem. Biophys. Res. Commun. 255, 328–333.

106. Mozley, D., Remberg, A., and Gartner, W. (1997)Large-scale generation of affinity-purified recombi-nant phytochrome chromopeptide. Photochem.Photobiol. 66, 710–715.

107. Kneip, C., Mozley, D., Hildebrandt, M. P., Gartner,W., Braslavsky, S. E., and Schaffner, K. (1997) Ef-fect of chromophore exchange on the resonanceRaman spectra of recombinant phytochromes. FEBSLett. 414, 23–26.

108. Remberg, A., Ruddat, A., Braslavsky, S. E., Gartner,W., and Schaffner, K. (1998) Chromophore incor-poration, Pr to Pfr kinetics, and Pfr thermal rever-sion of recombinant N-terminal fragments ofphytochrome A and B chromoproteins. Biochemis-try 37, 9983–9990.

109. Huecas, S., Villalba, M., Gonzalez, E., Martinez-Ruiz, A., and Rodriguez, R. (1999) Production anddetailed characterization of biologically active ol-ive pollen allergen Ole e 1 secreted by the yeastPichia pastoris. Eur. J. Biochem. 261, 539–546.

110. Ferrarese, L., Trainotti, L., Gattolin, S., andCasadoro, G. (1998) Secretion, purification and ac-tivity of two recombinant pepper endo-beta-1,4-glucanases expressed in the yeast Pichia pastoris.FEBS Lett. 422, 23–26.

111. Sowka, S., Hsieh, L. S., Krebitz, M., Akasawa, A.,Martin, B. M., Starrett, D., Peterbauer, C. K.,Scheiner, O., and Breiteneder, H. (1998) Identifica-tion and cloning of prs a 1, a 32-kDa endochitinaseand major allergen of avocado, and its expression inthe yeast Pichia pastoris. J. Biol. Chem. 273, 28091–28097.

112. Grobe, K., Becker, W.-M., Schlaak, M., Petersen, A.(1999) Grass group I allergens (beta-expansins) are

novel, papain-related proteinases. Eur. J. Biochem.263, 33–40.

113. Ruddat, A., Schmidt, P., Gatz, C., Braslavsky, S. E.,Gartner, W., and Schaffner, K. (1997) Recombinanttype A and B phytochromes from potato. Transientabsorption spectroscopy. Biochemistry 36, 103–111.

114. Hiller, K. M., Lubahn, B. C., and Klapper, D. G.(1998) Cloning and expression of ragweed allergenAmb a 6. Scand. J. Immunol. 48, 26–36.

115. Penheiter, A. R., Klucas, R. V., and Sarath, G. (1998)Purification and characterization of a soybean rootnodule phosphatase expressed in Pichia pastoris.Protein Expr. Purif. 14, 125–130.

116. Payne, M. S., Petrillo, K. L., Gavagan, J. E., Wagner,L. W., DiCosimo, R., and Anton, D. L. (1995) High-level production of spinach glycolate oxidase in themethylotrophic yeast Pichia pastoris: engineering abiocatalyst. Gene 167, 215–219.

117. Payne, M. S., Petrillo, K. L., Gavagan, J. E.,DiCosimo, R., Wagner, L. W., and Anton, D. L.(1997) Engineering Pichia pastoris for biocatalysis:co-production of two active enzymes. Gene 194,179–182.

118. Brandes, H. K., Hartman, F. C., Lu, T. Y., andLarimer, F. W. (1996) Efficient expression of thegene for spinach phosphoribulokinase in Pichiapastoris and utilization of the recombinant enzymeto explore the role of regulatory cysteinyl residuesby site-directed mutagenesis. J. Biol. Chem. 271,6490–6496.

119. Kristensen, A. K., Brunstedt, J., Nielsen, J. E.,Mikkelsen, J. D., Roepstorff, P., and Nielsen, K. K.(1999) Processing, disulfide pattern, and biologicalactivity of a sugar beet defensin, AX2, expressed inPichia pastoris. Protein Expr. Purif. 16, 377–387.

120. Petersen, A., Grobe, K., Lindner, B., Schlaak, M.,and Becker, W. M. (1997) Comparison of naturaland recombinant isoforms of grass pollen allergens.Electrophoresis 18, 819–825.

121. Brenner, E. D., Lambert, K. N., Kaloshian, I., andWilliamson, V. M. (1998) Characterization ofLeMir, a root-knot nematode-induced gene in to-mato with an encoded product secreted from theroot. Plant Physiol. 118, 237–247.

122. Klein, C., de Lamotte-Guery, F., Gautier, F., Mou-lin, G., Boze, H., Joudrier, P., and Gautier, M. F.(1998) High-level secretion of a wheat lipid transferprotein in Pichia pastoris. Protein Expr. Purif. 13,73–82.

123. Ogawa, M., Nakamura, S., Atsuchi, T., Tamiya, T.,Tsuchiya, T., and Nakai, S. (1999) Macromolecularantimicrobial glycoprotein, achacin, expressed in amethylotrophic yeast Pichia pastoris. FEBS Lett.448, 41–44.

124. Munshi, C. and Lee, H. C. (1997) High-level expres-sion of recombinant Aplysia ADP-ribosyl cyclase in

44 Cregg et al.


Pichia pastoris by fermentation. Protein Expr.Purif. 11, 104–110.

125. Monosov, E. Z., Wenzel, T. J., Luers, G. H., Heyman,J. A., and Subramani, S. (1996) Labeling of peroxi-somes with green fluorescent protein in living P.pastoris cells. J. Histochem. Cytochem. 44, 581–589.

126. Gallet, P. F., Vaujour, H., Petit, J. M., Maftah, A.,Oulmouden, A., Oriol, R., Le Narvor, C., Guilloton,M., and Julien, R. (1998) Heterologous expression ofan engineered truncated form of human Lewisfucosyltransferase (Fuc-TIII) by the methylotrophicyeast Pichia pastoris. Glycobiology 8, 919–925.

127. Garcia-Garcia, J. C., Soto, A., Nigro, F., Mazza, M.,Joglar, M., Hechevarria, M., Lamberti, J., and de laFuente, J. (1998) Adjuvant and immunostimulatingproperties of the recombinant Bm86 protein ex-pressed in Pichia pastoris. Vaccine 16, 1053–1055.

128. Garcia-Garcia, J. C., Montero, C., Rodriquez, M.,Soto, A., Redondo, M., Valdes, M., Mendez, L., andde la Fuente, J. (1998) Effect of particulation on theimmunogenic and protective properties of the recom-binant Bm86 antigen expressed in Pichia pastoris.Vaccine 16, 374–380.

129. Rodríguez, M., Rubiera, R., Penichet, M.,Montesinos, R., Cremata, J., Falcón, V., Sánchez, G.,Bringas, R., Cordovés, C., Valdés, M., Lleonart, R.,Herrera, L., and de la Fuente, J. (1994) High levelexpression of the B. microplus Bm86 antigen in theyeast Pichia pastoris forming highly immunogenicparticles for cattle. J. Biotechnol. 33, 135–146.

130. Vailes, L. D., Kinter, M. T., Arruda, L. K., andChapman, M. D. (1998) High-level expression ofcockroach allergen, Bla g 4, in Pichia pastoris. J. Al-lergy Clin. Immunol. 101, 274–280.

131. Williams, T. A., Michaud, A., Houard, X., Chauvet,M. T., Soubrier, F., and Corvol, P. (1996) Drosophilamelanogaster angiotensin I-converting enzyme ex-pressed in Pichia pastoris resembles the C domain ofthe mammalian homologue and does not requireglycosylation for secretion and enzymic activity.Biochem. J. 318, 125–131.

132. Jackson, V. N., Cameron, J. M., Zammit, V. A., andPrice, N. T. (1999) Sequencing and functional expres-sion of the malonyl-CoA-sensitive carnitinepalmitoyltransferase from Drosophila melanogaster.Biochem. J. 341, 483–489.

133. McCollum, D., Monosov, E., and Subramani, S.(1993) The pas8 mutant of Pichia pastoris exhibitsthe peroxisomal protein import deficiencies ofZellweger syndrome—the PAS8 protein binds to theCOOH-terminal tripeptide peroxisomal targeting sig-nal, and is a member of the TPR protein family. J.Cell Biol. 121, 761–774.

134. Canales, M., Enriquez, A., Ramos, E., Cabrera, D.,Dandie, H., Soto, A., Falcon, V., Rodriguez, M., andde la Fuente, J. (1997) Large-scale production in

Pichia pastoris of the recombinant vaccine Gavacagainst cattle tick. Vaccine 15, 414–422.

135. Brankamp, R. G., Sreekrishna, K., Smith, P. L.,Blankenship, D. T., and Cardin, A. D. (1995) Expres-sion of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropicyeast Pichia pastoris. Protein Expr. Purif. 6, 813–820.

136. Rosenfeld, S. A., Nadeau, D., Tirado, J., Hollis, G.F., Knabb, R. M., and Jia, S. (1996) Production andpurification of recombinant hirudin expressed in themethylotrophic yeast Pichia pastoris. Protein Expr.Purif. 8, 476–482.

137. Briand, L., Perez, V., Huet, J. C., Danty, E., Masson,C., and Pernollet, J. C. (1999) Optimization of theproduction of a honeybee odorant-binding protein byPichia pastoris. Protein Expr. Purif. 15, 362–369.

138. Danty, E., Briand, L., Michard-Vanhee, C., Perez, V.,Arnold, G., Gaudemer, O., Huet, D., Huet, J.-C.,Ouali, C., Masson, C., and Pernollet, J.-C. (1999)Cloning and expression of a queen pheromone-bind-ing protein in the honeybee: an olfactory-specific,developmentally regulated protein. J. Neuroscience19, 7468–7475.

139. Hussein, A. S., Chacon, M. R., Smith, A. M., Tosado-Acevedo, R., and Selkirk, M. E. (1999) Cloning, ex-pression, and properties of a nonneuronal secretedacetylcholinesterase from the parasitic nematodeNippostrongylus brasiliensis. J. Biol. Chem. 274,9312–9319.

140. Fahnestock, S. R. and Bedzyk, L. A. (1997) Produc-tion of synthetic spider dragline silk protein in Pichiapastoris. Appl. Microbiol. Biotechnol. 47, 33–39.

141. Monsalve, R. I., Lu, G., and King, T. P. (1999) Ex-pressions of recombinant venom allergen, antigen 5of yellowjacket (Vespula vulgaris) and paper wasp(Polistes annularis), in bacteria or yeast. ProteinExpr. Purif. 16, 410–416.

142. Vozza, L. A., Wittwer, L., Higgins, D. R., Purcell, T.J., Bergseid, M., Collins-Racie, L. A., LaVallie, E.R., and Hoeffler, J. P. (1996) Production of a recom-binant bovine enterokinase catalytic subunit in themethylotrophic yeast Pichia pastoris. Biotechnology(NY) 14, 77–81.

143. Samaddar, M., Catterall, J. F., and Dighe, R. R.(1997) Expression of biologically active beta subunitof bovine follicle-stimulating hormone in themethylotrophic yeast Pichia pastoris. Protein Expr.Purif. 10, 345–355.

144. Rodriguez, M., Martinez, V., Alazo, K., Suarez, M.,Redondo, M., Montero, C., Besada, V., and de laFuente, J. (1998) The bovine IFN-omega 1 is biologi-cally active and secreted at high levels in the yeastPichia pastoris. J. Biotechnol. 60, 3–14.

145. Digan, M. E., Lair, S. V., Brierley, R. A., Siegel, R.S., Williams, M. E., Ellis, S. B., Kellaris, P. A.,



Provow, S. A., Craig, W. S., Veliçelebi, G., Harpold,M. M., and Thill, G. P. (1989) Continuous produc-tion of a novel lysozyme via secretion from the yeast,Pichia pastoris. Biotechnology (NY) 7, 160–164.

146. Abdulaev, N. G., Popp, M. P., Smith, W. C., andRidge, K. D. (1997) Functional expression of bovineopsin in the methylotrophic yeast Pichia pastoris.Protein Expr. Purif. 10, 61–69.

147. Vedvick, T., Buckholtz, R. G., Engel, M., Urcan, M.,Kinney, J., Provow, S., Siegel, R. S., and Thill, G. P.(1991) High-level secretion of biologically activeaprotinin from the yeast Pichia pastoris. J. Ind.Microbiol. 7, 197–201.

148. Choi, B.-K. and Jimenez-Flores, R. (1996) Study ofputative glycosylation site in bovine beta-casein in-troduced by PCR-based site-directed mutagenesis. J.Agric. Food Chem. 44, 358–364.

149. Johnsen, L. B., Ravn, P., Berglund, L., Petersen, T.E., Rasmussen, L. K., Heegaard, C. W., Rasmussen,J. T., Benfeldt, C., and Fedosov, S. N. (1998) A re-fined kinetic analysis of plasminogen activation byrecombinant bovine tissue-type plasminogen activa-tor indicates two interconvertible activator forms.Biochemistry 37, 12631–12639.

150. Fedosov, S. N., Berglund, L., Nexo, E., and Petersen,T. E. (1999) Sequence, S-S bridges, and spectra ofbovine transcobalamin expressed in Pichia pastoris.J. Biol. Chem. 274, 26015–26020.

151. Wedlock, D. N., Goh, L. P., McCarthy, A. R., Mid-winter, R. G., Parlane, N. A., and Buddle, B. M.(1999) Physiological effects and adjuvanticity of re-combinant brushtail possum TNF-alpha. Immunol.Cell Biol. 77, 28–33.

152. Morel, N. and Massoulie, J. (1997) Expression andprocessing of vertebrate acetylcholinesterase in theyeast Pichia pastoris. Biochem. J. 328, 121–129.

153. Zhu, A., Monahan, C., Wang, Z. K., and Goldstein, J.(1996) Expression, purification, and characterizationof recombinant alpha-N-acetylgalactosaminidaseproduced in the yeast Pichia pastoris. Protein Expr.Purif. 8, 456–462.

154. Simon, S. and Massoulie, J. (1997) Cloning and ex-pression of acetylcholinesterase from Electrophorus.Splicing pattern of the 3' exons in vivo and in trans-fected mammalian cells. J. Biol. Chem. 272, 33045–33055.

155. Rautiainen, J., Auriola, S., Rouvinen, J., Kauppinen,J., Zeiler, T., Novikov, D., Virtanen, T., andMantyjarvi, R. A. (1998) Molecular and crystal prop-erties of Bos d 2, an allergenic protein of the lipocalinfamily. Biochem. Biophys. Res. Commun. 247, 746–750.

156. Weiss, H. M., Haase, W., Michel, H., and Reilander,H. (1998) Comparative biochemical and pharmaco-logical characterization of the mouse 5HT5A 5-hy-droxytryptamine receptor and the human beta

2-adrenergic receptor produced in the methylotrophicyeast Pichia pastoris. Biochem. J. 330, 1137–1147.

157. Werten, M. W., van den Bosch, T. J., Wind, R. D.,Mooibroek, H., and de Wolf, F. A. (1999) High-yieldsecretion of recombinant gelatins by Pichia pastoris.Yeast 15, 1087–1096.

158. Masure, S., Paemen, L., Van Aelst, I., Fiten, P.,Proost, P., Billiau, A., Van Damme, J., andOpdenakker, G. (1997) Production and characteriza-tion of recombinant active mouse gelatinase B fromeukaryotic cells and in vivo effects after intravenousadministration. Eur. J. Biochem. 244, 21–30.

159. Merkle, R. K., Zhang, Y., Ruest, P. J., Lal, A., Liao,Y. F., and Moremen, K. W. (1997) Cloning, expres-sion, purification, and characterization of the murinelysosomal acid alpha-mannosidase. Biochim.Biophys. Acta 1336, 132–146.

160. Ferrari, E., Lodi, T., Sorbi, R. T., Tirindelli, R.,Cavaggioni, A., and Spisni, A. (1997) Expression ofa lipocalin in Pichia pastoris: secretion, purificationand binding activity of a recombinant mouse majorurinary protein. FEBS Lett. 401, 73–77.

161. Urbatsch, I. L., Beaudet, L., Carrier, I., and Gros, P.(1998) Mutations in either nucleotide-binding site ofP-glycoprotein (Mdr3) prevent vanadate trapping ofnucleotide at both sites. Biochemistry 37, 4592–4602.

162. Beaudet, L., Urbatsch, I. L., and Gros, P. (1998)High-level expression of mouse Mdr3 P-glycopro-tein in yeast Pichia pastoris and characterization ofATPase activity. Methods Enzymol. 292, 397–413.

163. Beaudet, L., Urbatsch, I. L., and Gros, P. (1998) Mu-tations in the nucleotide-binding sites of P-glycopro-tein that affect substrate specificity modulatesubstrate-induced adenosine triphosphatase activity.Biochemistry 37, 9073–9082.

164. Eldin, P., Pauza, M. E., Hieda, Y., Lin, G.,Murtaugh, M. P., Pentel, P. R., and Pennell, C. A.(1997) High-level secretion of two antibody singlechain Fv fragments by Pichia pastoris. J. Immunol.Methods 201, 67–75.

165. Boehm, T., Pirie-Shepard, S., Trinh, L. B., Shiloach,J., and Folkman, J. (1999) Disruption of the KEX1gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast 15, 563–567.

166. Vallee, F., Lal, A., Moremen, K. W., and Howell, P.L. (1999) Purification, crystallization and prelimi-nary X-ray crystallographic analysis of recombinantmurine Golgi mannosidase IA, a class I alpha-mannosidase involved in Asn-linked oligosaccha-ride maturation. Acta Crystallogr. D Biol.Crystallogr. 55, 571–573.

167. Jerva, L. F., Sullivan, G., and Lolis, E. (1997) Func-tional and receptor binding characterization of recom-binant murine macrophage inflammatory protein 2:

46 Cregg et al.


sequence analysis and mutagenesis identify receptorbinding epitopes. Protein Sci. 6, 1643–1652.

168. Fidler, A. E., Lun, S., Young, W., and McNatty, K. P.(1998) Expression and secretion of a biologically ac-tive glycoprotein hormone, ovine follicle stimulatinghormone, by Pichia pastoris. J. Mol. Endocrinol. 21,327–336.

169. Ventura, S., Villegas, V., Sterner, J., Larson, J.,Vendrell, J., Hershberger, C. L., Aviles, F. X. (1999)Mapping the pro-region of carboxypeptidase B byprotein engineering. Cloning, overexpression, andmutagenesis of the porcine proenzyme. J. Biol. Chem.274, 19925–19933.

170. Richard, F., Robert, P., Remy, J. J., Martinat, N.,Bidart, J. M., Salesse, R., and Combarnous, Y. (1998)High-level secretion of biologically active recombi-nant porcine follicle-stimulating hormone by themethylotrophic yeast Pichia pastoris. Biochem.Biophys. Res. Commun. 245, 847–852.

171. Wuebbens, M. W., Roush, E. D., Decastro, C. M., andFierke, C. A. (1997) Cloning, sequencing, and recom-binant expression of the porcine inhibitor of carbonicanhydrase: a novel member of the transferrin family.Biochemistry 36, 4327–4336.

172. Reddy, R. G., Yoshimoto, T., Yamamoto, S., andMarnett, L. J. (1994) Expression, purification, andcharacterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast,Pichia pastoris. Biochem. Biophys. Res. Commun.205, 381–388.

173. Doring, F., Theis, S., and Daniel, H. (1997) Expres-sion and functional characterization of the mamma-lian intestinal peptide transporter PEPT1 in themethylotrophic yeast Pichia pastoris. Biochem.Biophys. Res. Commun. 232, 656–662.

174. Doring, F., Michel, T., Rosel, A., Nickolaus, M., andDaniel, H. (1998) Expression of the mammalian re-nal peptide transporter PEPT2 in the yeast Pichiapastoris and applications of the yeast system for func-tional analysis. Mol. Membr. Biol. 15, 79–88.

175. Ridder, R., Schmitz, R., Legay, F., and Gram, H.(1995) Generation of rabbit monoclonal antibodyfragments from a combinatorial phage display libraryand their production in the yeast Pichia pastoris. Bio-technology (NY) 13, 255–260.

176. Kotake, H., Li, Q., Ohnishi, T., Ko, K. W.,Agellon, L. B., and Yokoyama, S. (1996) Expres-sion and secretion of rabbit plasma cholesteryl es-ter transfer protein by Pichia pastoris. J. Lipid Res.37, 599–605.

177. Sadhukhan, R., Sen, G. C., and Sen, I. (1996) Syn-thesis and cleavage—secretion of enzymatically ac-tive rabbit angiotensin-converting enzyme in Pichiapastoris. J. Biol. Chem. 271, 18310–18313.

178. Heim, J., Schmidt-Dannert, C., Atomi, H., andSchmid, R. D. (1998) Functional expression of amammalian acetylcholinesterase in Pichia pastoris:

comparison to acetylcholinesterase, expressed andreconstituted from Escherichia coli. Biochim.Biophys. Acta 1396, 306–319.

179. He, C., Alexander, J. J., Lim, A., and Quigg, R. J.(1997) Production of the rat complement regulator,Crry, as an active soluble protein in Pichia pastoris.Arch. Biochem. Biophys. 341, 347–352.

180. Chen, Y. J. and Gonatas, N. K. (1997) The Golgisialoglycoprotein MG160, expressed in Pichiapastoris, does not require complex carbohydrates andsialic acid for secretion and basic fibroblast growthfactor binding. Biochem. Biophys. Res. Commun.234, 68–72.

181. Mistry, A. R., Falciola, L., Monaco, L., Tagliabue,R., Acerbis, G., Knight, A., Harbottle, R. P., Soria,M., Bianchi, M. E., Coutelle, C., and Hart, S. L.(1997) Recombinant HMG1 protein produced inPichia pastoris: a nonviral gene delivery agent.Biotechniques 22, 718–729.

182. Zhu, H., Shi, J., de Vries, Y., Arvidson, D. N., Cregg,J. M., and Woldegiorgis, G. (1997) Functional stud-ies of yeast-expressed human heart muscle carnitinepalmitoyltransferase I. Arch. Biochem. Biophys. 347,53–61.

183. de Vries, Y., Arvidson, D. N., Waterham, H. R.,Cregg, J. M., and Woldegiorgis, G. (1997) Functionalcharacterization of mitochondrial carnitinepalmitoyltransferases I and II expressed in the yeastPichia pastoris. Biochemistry 36, 5285–5292.

184. Kasper, C, Rasmussen, H., Berezin, V., Bock, E., andLarsen, I. K. (1999) Expression, crystallization andpreliminary X-ray analysis of the two amino-termi-nal Ig domains of the neural cell adhesion molecule(NCAM). Acta Crystallogr. D Biol. Crystallogr. 55,1598–1600.

185. Gachhui, R., Presta, A., Bentley, D. F., Abu-Soud, H.M., McArthur, R., Brudvig, G., Ghosh, D. K., andStuehr, D. J. (1996) Characterization of the reductasedomain of rat neuronal nitric oxide synthase gener-ated in the methylotrophic yeast Pichia pastoris.Calmodulin response is complete within the reduc-tase domain itself. J. Biol. Chem. 271, 20594–20602.

186. Qin, Y. M., Poutanen, M. H., Helander, H. M., Kvist,A. P., Siivari, K. M., Schmitz, W., Conzelmann, E.,Hellman, U., and Hiltunen, J. K. (1997) Peroxisomalmultifunctional enzyme of beta-oxidation metaboliz-ing D-3-hydroxyacyl-CoA esters in rat liver: molecu-lar cloning, expression and characterization.Biochem. J. 321, 21–28.

187. Yu, Y., Vranken, W., Goudreau, N., de Miguel, E.,Magny, M. C., Mort, J. S., Dupras, R., Storer, A. C.,and Ni, F. (1998) An NMR-based identification ofpeptide fragments mimicking the interactions of thecathepsin B propeptide. FEBS Lett. 429, 9–16.

188. Sivaraman, J., Coulombe, R., and Cygler, M. (1996)Crystallization of rat procathepsin B. ActaCrystallogr. 52, 874.



189. Loewen, M. C., Liu, X., Davies, P. L., and Daugulis,A. J. (1997) Biosynthetic production of type II fishantifreeze protein: fermentation by Pichia pastoris.Appl. Microbiol. Biotechnol. 48, 480–486.

190. Trant, J. M. (1996) Functional expression of recom-binant spiny dogfish shark (Squalus acanthias) cyto-chrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris). Arch.Biochem. Biophys. 326, 8–14.

191. Weiss, S., Famulok, M., Edenhofer, F., Wang, Y.-H.,Jones, I. M., Groschup, M., and Winnacker, E. L.(1995) Overexpression of active Syrian goldenhamster prion protein PrPc as a glutathione S-trans-ferase fusion in heterologous systems. J. Virol. 69,4776–4783.

192. Tremblay, L. O., Campbell Dyke, N., and Herscovics,A. (1998) Molecular cloning, chromosomal mappingand tissue-specific expression of a novel human al-pha1,2-mannosidase gene involved in N-glycanmaturation. Glycobiology 8, 585–595.

193. Zhu, A., Wang, Z. K., and Beavis, R. (1998) Struc-tural studies of alpha-N-acetylgalactosaminidase: ef-fect of glycosylation on the level of expression,secretion efficiency, and enzyme activity. Arch.Biochem. Biophys. 352, 1–8.

194. Kang, H. A., Sohn, J. H., Choi, E. S., Chung, B. H.,Yu, M. H., and Rhee, S. K. (1998) Glycosylation ofhuman alpha 1-antitrypsin in Saccharomycescerevisiae and methylotrophic yeasts. Yeast 14, 371–381.

195. Lee, K. N., Tae, W.-C., Jackson, K. W., Kwon, S. H.,and McKee, P. A. (1999) Characterization of wild-type and mutant alpha 2-antiplasmins: fibrinolysis en-hancement by reactive site mutant. Blood 94,164–171.

196. Talmont, F., Sidobre, S., Demange, P., Milon, A., andEmorine, L. J. (1996) Expression and pharmacologi-cal characterization of the human -opioid receptorin the methylotrophic yeast Pichia pastoris. FEBSLett. 394, 268–272.

197. O’Connell, M. A., Gerber, A., and Keegan, L. P.(1998) Purification of native and recombinantdouble-stranded RNA-specific adenosine deami-nases. Methods 15, 51–62.

198. Le Brocque, D., Henry, A., Cappai, R., Li, Q. X.,Tanner, J. E., Galatis, D., Gray, C., Holmes, S.,Underwood, J. R., Beyreuther, K., Masters, C. L., andEvin, G. (1998) Processing of the Alzheimer’s dis-ease amyloid precursor protein in Pichia pastoris:immunodetection of alpha-, beta-, and gamma-secretase products. Biochemistry 37, 14958–14965.

199. Mok, S. S., Sberna, G., Heffernan, D., Cappai, R.,Galatis, D., Clarris, H. J., Sawyer, W. H., Beyreuther,K., Masters, C. L., and Small, D. H. (1997) Expres-sion and analysis of heparin-binding regions of theamyloid precursor protein of Alzheimer’s disease.FEBS Lett. 415, 303–307.

200. Cappai, R., Mok, S. S., Galatis, D., Tucker, D. F.,Henry, A., Beyreuther, K., Small, D. H. and Masters,C. L. (1999) Recombinant human amyloid precursor-like protein 2 (APLP2) expressed in the yeast Pichiapastoris can stimulate neurite outgrowth. FEBS Lett.442, 95–98.

201. Henry, A., Masters, C. L., Beyreuther, K., andCappai, R. (1997) Expression of human amyloid pre-cursor protein ectodomains in Pichia pastoris: analy-sis of culture conditions, purification, andcharacterization. Protein Expr. Purif. 10, 283–291.

202. Culvenor, J. G., Henry, A., Hartmann, T., Evin, G.,Galatis, D., Friedhuber, A., Jayasena, U. L.,Underwood, J. R., Beyreuther, K., Masters, C. L., andCappai, R. (1998) Subcellular localization of theAlzheimer’s disease amyloid precursor protein andderived polypeptides expressed in a recombinantyeast system. Amyloid 5, 79–89.

203. Markaryan, A., Morosova, I., Lee, B.-S., and Kaplan,A. (1999) Atypical processing of amyloid precursorfusion protein by proteolytic activity in Pichiapastoris. Biochem. Biophys. Res. Commun. 262, 263–268.

204. Ohsawa, I., Hirose, Y., Ishiguro, M., Imai, Y., Ishiura,S., and Kohsaka, S. (1995) Expression, purification,and neurotrophic activity of amyloid precursor pro-tein-secreted forms produced by yeast. Biochem.Biophys. Res. Commun. 213, 52–58.

205. Sahasrabudhe, A. V., Solapure, S. M., Khurana, R.,Suryanarayan, V., Ravishankar, S., deSousa, S. M.,and Das, G. (1998) Production of recombinant hu-man bile salt stimulated lipase and its variant inPichia pastoris. Protein Expr. Purif. 14, 425–433.

206. FitzGerald, K., Hollinger, P., and Winter, G. (1997)Improved tumour targeting by disulphide stabilizeddiabodies expressed in Pichia pastoris. Protein Eng.10, 1221–1225.

207. Lam, L. P. and Berger, S. A. (1997) Intracellular ex-pression and purification of the c-kit receptor kinasedomain in Pichia pastoris. Biotechniques 23, 83–86.

208. Lam, L. P., Chow, R. Y., and Berger, S. A. (1999) Atransforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain. Biochem. J. 338,131–138.

209. You, Y. H., Hefta, L. J., Yazaki, P. J., Wu, A. M., andShively, J. E. (1998) Expression, purification, andcharacterization of a two domain carcinoembryonicantigen minigene (N-A3) in Pichia pastoris. The es-sential role of the N-domain. Anticancer Res. 18,3193–3201.

210. Sun, J., Bottomley, S. P., Kumar, S., and Bird, P. I.(1997) Recombinant caspase-3 expressed in Pichiapastoris is fully activated and kinetically indistin-guishable from the native enzyme. Biochem. Biophys.Res. Commun. 238, 920–924.

211. Linnevers, C. J., McGrath, M. E., Armstrong, R.,Mistry, F. R., Barnes, M. G., Klaus, J. L., Palmer, J.

48 Cregg et al.


T., Katz, B. A., and Bromme, D. (1997) Expressionof human cathepsin K in Pichia pastoris and prelimi-nary crystallographic studies of an inhibitor complex.Protein Sci. 6, 919–921.

212. Hou, W. S., Brommer, D., Zhao, Y., Mehler, E.,Dushey, C., Weinstein, H., Miranda, C. S., Fraga, C.,Greig, F., Carey, J., Rimoin, D. L., Desnick, R. J.,and Gelb, B. D. (1999) Characterization of novelcathepsin K mutations in the pro and mature polypep-tide regions causing pycnodysostosis. J. Clin. Invest.103, 731–738.

213. Menard, R., Carmona, E., Takebe, S., Dufour, E.,Plouffe, C., Mason, P., and Mort, J. S. (1998) Auto-catalytic processing of recombinant humanprocathepsin L. Contribution of both intermolecularand unimolecular events in the processing ofprocathepsin L in vitro. J. Biol. Chem. 273, 4478–4484.

214. Carmona, E., Dufour, E., Plouffe, C., Takebe, S.,Mason, P., Mort, J. S., and Menard, R. (1996) Po-tency and selectivity of the cathepsin L propeptide asan inhibitor of cysteine proteases. Biochemistry 35,8149–8157.

215. Bromme, D., Li, Z., Barnes, M., and Mehler, E.(1999) Human cathepsin V functional expression, tis-sue distribution, electrostatic surface potential, enzy-matic characterization, and chromosomallocalization. Biochemistry 38, 2377–2385.

216. Nägler, D. K., Zhang, R., Tam, W., Sulea, T.,Purisima, E. O., and Menard, R. (1999) Human cathe-psin X: a cysteine protease with unique carboxypep-tidase activity. Biochemistry 38, 12648–12654.

217. Munshi, C. B., Fryxell, K. B., Lee, H. C., andBranton, W. D. (1997) Large-scale production of hu-man CD38 in yeast by fermentation. MethodsEnzymol. 280, 318–330.

218. McGrew, J. T., Leiske, D., Dell, B., Klinke, R.,Krasts, D., Wee, S. F., Abbott, N., Armitage, R., andHarrington, K. (1997) Expression of trimeric CD40ligand in Pichia pastoris: use of a rapid method todetect high-level expressing transformants. Gene 187,193–200.

219. Gerstmayer, B., Altenschmidt, U., Hoffmann, M., andWels, W. (1997) Costimulation of T cell prolifera-tion by a chimeric B7-2 antibody fusion protein spe-cifically targeted to cells expressing the erbB2proto-oncogene. J. Immunol. 158, 4584–4590.

220. Sen Gupta, C. and Dighe, R. R. (1999)Hyperexpression of biologically active human chori-onic gonadotropin using the methylotropic yeastPichia pastoris. J. Mol. Endocrinol. 22, 273–283.

221. Daniels, G. L., Green, C. A., Powell, R. M., andWard, T. (1998) Hemagglutination inhibition ofCromer blood group antibodies with soluble recom-binant decay-accelerating factor. Transfusion 38,332–336.

222. Battista, M. C., Bergamini, G., Campanini, F.,Landini, M. P., and Ripalti, A. (1996) Intracellularproduction of a major cytomegalovirus antigenic pro-tein in the methylotrophic yeast Pichia pastoris. Gene176, 197–201.

223. Powell, R. M., Ward, T., Evans, D. J., and Almond, J.W. (1997) Interaction between echovirus 7 and itsreceptor, decay-accelerating factor (CD55): evidencefor a secondary cellular factor in A-particle forma-tion. J. Virol. 71, 9306–9312.

224. Gerber, A., O’Connell, M. A., and Keller, W. (1997)Two forms of human double-stranded RNA-specificeditase 1 (hRED1) generated by the insertion of anAlu cassette. RNA 3, 453–463.

225. Boehm, T., O’Reilly, M. S., Keough, K., Shiloach,J., Shapiro, R., and Folkman, J. (1998) Zinc–bind-ing of endostatin is essential for its angiogenic ac-tivity. Biochem. Biophys. Res. Commun. 252,190–194.

226. Zhan, H., Liu, B., Reid, S. W., Aoki, K. H., Li, C.,Syed, R. S., Karkaria, C., Koe, G., Sitney, K.,Hayenga, K., Mistry, F., Savel, L., Dreyer, M.,Katz, B. A., Schreurs, J., Matthews, D. J.,Cheetham, J. C., Egrie, J., Giebel, L., and Stroud,R. M. (1999) Engineering a soluble extracellularerythropoietin receptor (EPObp) in Pichia pastoristo eliminate microheterogeneity, and its complexwith erythropoietin. Protein Eng. 12, 505–513.

227. Tanaka, M., Suda, T., Yatomi, T., Nakamura, N.,and Nagata, S. (1997) Lethal effect of recombinanthuman Fas ligand in mice pretreated with Propi-onibacterium acnes. J. Immunol. 158, 2303–2309.

228. Cote, H. C., Pratt, K. P., Davie, E. W., and Chung,D. W. (1997) The polymerization pocket “a” withinthe carboxy-terminal region of the gamma chain ofhuman fibrinogen is adjacent to but independentfrom the calcium-binding site. J. Biol. Chem. 272,23792–23798.

229. Rosenfeld, S. A., Ross, O. H., Hillman, M. C.,Corman, J. I., and Dowling, R. L. (1996) Productionand purification of human fibroblast collagenase(MMP-1) expressed in the methylotrophic yeastPichia pastoris. Protein Expr. Purif. 7, 423–430.

230. Spraggon, G., Applegate, D., Everse, S. J., Zhang,J. Z., Veerapandian, L., Redman, C., D o o l i t t l e ,R. F., and Grieninger, G. (1998) Crystal structure ofa recombinant alpha EC domain from human fi-brinogen-420. Proc. Natl. Acad. Sci. U.S.A. 95,9099–9104.

231. Yamada, M., Azuma, T., Matsuba, T., Iida, H.,Suzuki, H., Yamamoto, K., Kohli, Y., and Hori, H.(1994) Secretion of human intracellular aspartic pro-teinase cathepsin E expressed in the methylotrophicyeast, Pichia pastoris and characterization of pro-duced recombinant cathepsin E. Biochim. Biophys.Acta 1206, 279–285.



232. Roy, N., Padmanabhan, S., Smith, M., Shi, L., Navre,M., and Das, G. (1999) Expression of humangelatinase B in Pichia pastoris. Protein Expr. Purif.16, 324–330.

233. Sun, J., Bird, C. H., Buzza, M. S., McKee, K. E.,Whisstock, J. C., and Bird, P. I. (1999) Expressionand purification of recombinant human granzyme Bfrom Pichia pastoris. Biochem. Biophys. Res.Commun. 261, 251–255.

234. Zhu, H., Shi, J., Cregg, J. M., and Woldegiorgis, G.(1997) Reconstitution of highly expressed humanheart muscle carnitine palmitoyltransferase I.Biochem. Biophys. Res. Commun. 239, 498–502.

235. Kjeldsen, T., Pettersson, A. F., and Hach, M. (1999)Secretory expression and characterization of insulinin Pichia pastoris. Biotechnol. Appl. Biochem. 29,79–86.

236. Brierley, R. A. (1998) Secretion of recombinant hu-man insulin-like growth factor I (IGF-1). MethodsMol. Biol. 103, 149–177.

237. Green, M. M., Larkin, J., III, Subramaniam, P. S.,Szente, B. E., and Johnson, H. M. (1998) Human IFNgamma receptor cytoplasmic domain: expression andinteraction with HuIFN gamma. Biochem. Biophys.Res. Commun. 243, 170–176.

238. Johnson, T. M., Holaday, S. K., Sun, Y.,Subramaniam, P. S., Johnson, H. M., and Krishna, N.R. (1999) Expression, purification, and characteriza-tion of interferon-tau produced in Pichia pastorisgrown in a minimal medium. J. Interferon CytokineRes. 19, 631–636.

239. Murphy, K. P., Jr., Gagne, P., Pazmany, C., andMoody, M. D. (1998) Expression of humaninterleukin-17 in Pichia pastoris: purification andcharacterization. Protein Expr. Purif. 12, 208–214.

240. Sun, J., Coughlin, P., Salem, H. H., and Bird, P.(1995) Production and characterization of recombi-nant human proteinase inhibitor 6 expressed in Pichiapastoris. Biochim. Biophys. Acta 1252, 28–34.

241. Wagner, S. L., Siegel, R. S., Vedvick, T. S., Raschke,W. C., and Van Nostrand, W. E. (1992) High levelexpression, purification, and characterization of theKunitz-type protease inhibitor domain of proteasenexin-2/amyloid beta-protein precursor. Biochem.Biophys. Res. Commun. 186, 1138–1145.

242. Zhang, J. G., Owczarek, C. M., Ward, L. D., Howlett,G. J., Fabri, L. J., Roberts, B. A., and Nicola, N. A.(1997) Evidence for the formation of a heterotrimericcomplex of leukaemia inhibitory factor with its re-ceptor subunits in solution. Biochem. J. 325, 693–700.

243. Fryxell, K. B., O’Donoghue, K., Graeff, R. M., Lee,H. C., and Branton, W. D. (1995) Functional expres-sion of soluble forms of human CD38 in Escherichiacoli and Pichia pastoris. Protein Expr. Purif. 6, 329–336.

244. Liao, Y. F., Lal, A., and Moremen, K. W. (1996)Cloning, expression, purification, and characteriza-tion of the human broad specificity lysosomal acidalpha-mannosidase. J. Biol. Chem. 271, 28348–28358.

245. Chan, H., Elrod, K. C., Numerof, R. P., Sideris, S.,and Clark, J. M. (1999) Expression and characteriza-tion of recombinant mast cell tryptase. Protein Expr.Purif. 15, 251–257.

246. Niles, A. L., Maffitt, M., Haak-Frendscho, M.,Wheeless, C. J., and Johnson, D. A. (1998) Recombi-nant human mast cell tryptase beta: stable expressionin Pichia pastoris and purification of fully active en-zyme. Biotechnol. Appl. Biochem. 28, 125–131.

247. Kalandadze, A., Galleno, M., Foncerrada, L.,Strominger, J. L., and Wucherpfennig, K. W. (1996)Expression of recombinant HLA-DR2 molecules.Replacement of the hydrophobic transmembrane re-gion by a leucine zipper dimerization motif allowsthe assembly and secretion of soluble DR alpha-betaheterodimers. J. Biol. Chem. 271, 20156–20162.

248. Luo, D., Mah, N., Krantz, M., Wilde, K., Wishart, D.,Zhang, Y., Jacobs, F., and Martin, L. (1995) Vl-linker-Vh orientation-dependent expression of singlechain Fv-containing an engineered disulfide-stabi-lized bond in the framework regions. J. Biochem. (To-kyo) 118, 825–831.

249. Beall, C. J., Breckenridge, S. M., Chakravarty, L., andKolattukudy, P. E. (1998) Expression of humanmonocyte chemoattractant protein-1 in the yeastPichia pastoris. Protein Expr. Purif. 12, 145–150.

250. Masure, S., Paemen, L., Proost, P., Van Damme, J.,and Opdenakker, G. (1995) Expression of a humanmutant monocyte chemotactic protein 3 in Pichiapastoris and characterization as an MCP-3 receptorantagonist. J. Interferon Cytokine Res. 15, 955–963.

251. Kroll, K. A., Otte, S., Hirschfeld, G., Barnikol-Watanabe, S., Gotz, H., Sternbach, H., Kratzin, H. D.,Barnikol, H. U., and Hilschmann, N. (1999) Heter-ologous overexpression of human NEFA and studieson the two EF-hand calcium-binding sites. Biochem.Biophys. Res. Commun. 260, 1–8.

252. Kiselyov, V. V., Berezin, V., Maar, T. E., Soroka, V.,Edvardsen, K., Schousboe, A., and Bock, E. (1997)The first immunoglobulin-like neural cell adhesionmolecule (NCAM) domain is involved in double-re-ciprocal interaction with the second immunoglobu-lin-like NCAM domain and in heparin binding. J.Biol. Chem. 272, 10125–10134.

253. Yang, Y. S., Yang, M. C., Tucker, P. W., and Capra,J. D. (1997) NonO enhances the association of manyDNA-binding proteins to their targets. Nucleic AcidsRes. 25, 2284–2292.

254. Moritz, R. L., Ward, L. D., Tu, G.-F., Fabri, L. J., Ji,H., Yasukawa, K., and Simpson, R. J. (1999) The N-terminus of gp130 is critical for the formation of the

50 Cregg et al.


high-affinity interleukin-6 receptor complex. GrowthFactors 16, 265–278.

255. Rydberg, E. H., Sidhu, G., Vo, H. C., Hewitt, J., Cote,H. C., Wang, Y., Numao, S., MacGillivray, R. T.,Overall, C. M., Brayer, G. D., and Withers, S. G.(1999) Cloning, mutagenesis, and structural analysisof human pancreatic alpha-amylase expressed inPichia pastoris. Protein Sci. 8, 635–643.

256. Yang, Y. and Lowe, M.E. (1998) Human pancreatictriglyceride lipase expressed in yeast cells: purificationand characterization. Protein Expr. Purif. 13, 36–40.

257. Dufour, E., Tam, W., Nagler, D. K., Storer, A. C.,and Menard, R. (1998) Synthesis of amidrazones us-ing an engineered papain nitrile hydratase. FEBS Lett.433, 78–82.

258. Heimo, H., Palmu, K., and Suominen, I. (1998) Hu-man placental alkaline phosphatase: expression inPichia pastoris, purification and characterization ofthe enzyme. Protein Expr. Purif. 12, 85–92.

259. Dutta, B., Mukhopadhyay, D., Roy, N., Das, G., andKarande, A. A. (1998) Cloning, expression, purifica-tion, and immunocharacterization of placental pro-tein-14. Protein Expr. Purif. 14, 327–334.

260. Duman, J. G., Miele, R. G., Liang, H., Grella, D. K.,Sim, K. L., Castellino, F. J., and Bretthauer, R. K.(1998) O-Mannosylation of Pichia pastoris cellularand recombinant proteins. Biotechnol. Appl.Biochem. 28, 39–45.

261. Sim, B. K., O’Reilly, M. S., Liang, H., Fortier, A. H.,He, W., Madsen, J. W., Lapcevich, R., and Nacy, C.A. (1997) A recombinant human angiostatin proteininhibits experimental primary and metastatic cancer.Cancer Res. 57, 1329–1334.

262. Nilsen S. L., Prorok, M., Castellino, F. J. (1999) En-hancement through mutagenesis of the binding of theisolated kringle 2 domain of human plasminogen toomega-amino acid ligands and to an internal sequenceof a Streptococcal surface protein. J. Biol. Chem. 274,22380–22386.

263. Reverter, D., Ventura, S., Villegas, V., Vendrell, J.,and Aviles, F. X. (1998) Overexpression of humanprocarboxypeptidase A2 in Pichia pastoris and de-tailed characterization of its activation pathway. J.Biol. Chem. 273, 3535–3541.

264. Illy, C., Quraishi, O., Wang, J., Purisima, E., Vernet,T., and Mort, J. S. (1997) Role of occluding loop incathepsin B activity. J. Biol. Chem. 272, 1197–1202.

265. Cordle, R. A. and Lowe, M. E. (1998) Purificationand characterization of human procolipase expressedin yeast cells. Protein Expr. Purif. 13, 30–35.

266. Lima, C. D., Klein, M. G., Weinstein, I. B., andHendrickson, W. A. (1996) Three-dimensional struc-ture of human protein kinase C interacting protein 1,a member of the HIT family of proteins. Proc. Natl.Acad. Sci. U.S.A. 93, 5357–5362.

267. Harmsen, M. C., Heeringa, P., van der Geld, Y. M.,Huitema, M. G., Klimp, A., Tiran, A., and

Kallenberg, C. G. (1997) Recombinant proteinase 3(Wegener’s antigen) expressed in Pichia pastoris isfunctionally active and is recognized by patient sera.Clin. Exp. Immunol. 110, 257–264.

268. Dahlen, J. R., Foster, D. C., and Kisiel, W. (1997) Ex-pression, purification, and inhibitory properties of humanproteinase inhibitor. Biochemistry 36, 14874–14882.

269. Luo, D., Geng, M., Schultes, B., Ma, J., Xu, D. Z.,Hamza, N., Qi, W., Noujaim, A. A., andMadiyalakan, R. (1998) Expression of a fusion pro-tein of scFv-biotin mimetic peptide for immunoassay.J. Biotechnol. 65, 225–228.

270. Pennell, C. A. and Eldin, P. (1998) In vitro produc-tion of recombinant antibody fragments in Pichiapastoris. Res. Immunol. 149, 599–603.

271. Ohtani, W., Nawa, Y., Takeshima, K., Kamuro, H.,Kobayashi, K., and Ohmura, T. (1998) Physico-chemical and immunochemical properties of recom-binant human serum albumin from Pichia pastoris.Anal. Biochem. 256, 56–62.

272. Ikegaya, K., Hirose, M., Ohmura, T., and Nokihara,K. (1997) Complete determination of disulfide formsof purified recombinant human serum albumin, se-creted by the yeast Pichia pastoris. Anal. Chem. 69,1986–1991.

273. Ohtani, W., Ohda, T., Sumi, A., Kobayashi, K., andOhmura, T. (1998) Analysis of Pichia pastoris com-ponents in recombinant human serum albumin by im-munological assays and by HPLC with pulsedamperometric detection. Anal. Chem. 70, 425–429.

274. Petersen, C. E., Ha, C.-E., Harohalli, K., Park, D. S.,Feix, J. B., Isozaki, O., and Bhagavan, N. V. (1999)Structural investigations of a new familialdysalbuminemic hyperthyroxinemia genotype. Clin.Chem. 45, 1248–1254.

275. Steinlein, L. M., Graf, T. N., and Ikeda, R. A. (1995)Production and purification of N-terminal half-transfer-rin in Pichia pastoris. Protein Expr. Purif. 6, 619–624.

276. Bewley, M. C., Tam, B. M., Grewal, J., He, S., Shewry,S., Murphy, M. E., Mason, A. B., Woodworth, R. C.,Baker, E. N., and MacGillivray, R. T. (1999) X-raycrystallography and mass spectroscopy reveal that theN-lobe of human transferrin expressed in Pichiapastoris is folded correctly but is glycosylated onserine-32. Biochemistry 38, 2535–2541.

277. Mason, A. B., Woodworth, R. C., Oliver, R. W. A.,Green, B. N., Lin, L. N., Brandts, J. F., Tam, B. M.,Maxwell, A., and MacGillivray, R. T. (1996) Produc-tion and isolation of the recombinant N-lobe of hu-man serum transferrin from the methylotrophic yeastPichia pastoris. Protein Expr. Purif. 8, 119–125.

278. Sui, L. M., Lennon, J., Ma, C., McCann, I., Woo, I.,and Petra, P. H. (1999) Heterologous expression ofwild type and deglycosylated human sex steroid-binding protein (SBP or SHBG) in the yeast, Pichiapastoris. Characterization of the recombinant pro-teins. J. Steroid Biochem. Mol. Biol. 68, 119–127.



279. Tsujikawa, M., Okabayashi, K., Morita, M., andTanabe, T. (1996) Secretion of a variant of humansingle-chain urokinase-type plasminogen activatorwithout an N-glycosylation site in the methylotrophicyeast, Pichia pastoris and characterization of the se-creted product. Yeast 12, 541–553.

280. White, C. E., Hunter, M. J., Meininger, D. P., White, L.R., and Komives, E. A. (1995) Large-scale expression,purification and characterization of small fragments ofthrombomodulin: the roles of the sixth domain and ofmethionine 388. Protein Eng. 8, 1177–1187.

281. Austin, A. J., Jones, C. E., and Heeke, G. V. (1998)Production of human tissue factor using the Pichiapastoris expression system. Protein Expr. Purif. 13,136–142.

282. Chan, H., Springmann, E. B., and Clark, J. M. (1998)Expression and characterization of human tissue kal-likrein variants. Protein Expr. Purif. 12, 361–370.

283. Katz, B. A., Liu, B., Barnes, M., and Springman, E. B.(1998) Crystal structure of recombinant human tissuekallikrein at 2.0 Å resolution. Protein Sci. 7, 875–885.

284. Cregg, J. M. and Madden, K. R. (1989) Use of site-specific recombination to regenerate selectable mark-ers. Mol. Gen. Genet. 219, 320–323.

285. Miele, R. G., Castellino, F. J., and Bretthauer, R. K.(1997) Characterization of the acidic oligosaccharidesassembled on the Pichia pastoris-expressed recombi-nant kringle 2 domain of human tissue-type plasmino-gen activator. Biotechnol. Appl. Biochem. 26, 79–83.

286. Miele, R. G., Nilsen, S. L., Brito, T., Bretthauer, R.K., and Castellino, F. J. (1997) Glycosylation proper-ties of the Pichia pastoris-expressed recombinantkringle 2 domain of tissue-type plasminogen activa-tor. Biotechnol. Appl. Biochem. 25, 151–157.

287. Chang, T., Zajicek, J., and Castellino, F. J. (1997)Role of tryptophan-63 of the kringle 2 domain of tis-sue-type plasminogen activator in its thermal stabil-ity, folding, and ligand binding properties.Biochemistry 36, 7652–7663.

288. Glansbeek, H. L., van Beuningen, H. M., Vitters, E.L., van der Kraan, P. M., and van den Berg, W. B.(1998) Expression of recombinant human solubletype II transforming growth factor-beta receptor inPichia pastoris and Escherichia coli: two powerfulsystems to express a potent inhibitor of transforminggrowth factor-beta. Protein Expr. Purif. 12, 201–207.

289. Sreekrishna, K., Potenz, R. H. B., Cruze, J. A.,McCombie, W. R., Parker, K. A., Nelles, L.,Mazzaferro, P. K., Holden, K. A., Harrison, R. G.,Wood, P. J., Phelps, D. A., Hubbard, C. E., and Fuke,M. (1988) High level expression of heterologous pro-teins in methylotrophic yeast Pichia pastoris. J. Ba-sic Microbiol. 28, 265–278.

290. Rodenburg, K. W., Kjoller, L., Petersen, H. H., andAndreasen, P. A. (1998) Binding of urokinase-typeplasminogen activator-plasminogen activator inhibi-tor-1 complex to the endocytosis receptors alpha 2-

macroglobulin receptor/low-density lipoprotein re-ceptor-related protein and very-low-density lipopro-tein receptor involves basic residues in the inhibitor.Biochem. J. 329, 55–63.

291. Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi,T., and Kivirikko, K. I. (1997) Assembly of humanprolyl 4-hydroxylase and type III collagen in the yeastPichia pastoris: formation of a stable enzyme tet-ramer requires coexpression with collagen and as-sembly of a stable collagen requires coexpressionwith prolyl 4-hydroxylase. EMBO J. 16, 6702–6712.

292. Okabayashi, K., Tsujikawa, M., Morita, M., Einaga,K., Tanaka, K., Tanabe, T., Yamanouchi, K., Hirama,M., Tait, J. F., and Fujikawa, K. (1996) Secretory pro-duction of recombinant urokinase-type plasminogenactivator-annexin V chimeras in Pichia pastoris.Gene 177, 69–76.

293. Mohanraj, D., Olson, T., and Ramakrishnan, S. (1995)Expression of biologically active human vascular endot-helial growth factor in yeast. Growth Factors 12, 17–27.

294. Saelens, X., Vanlandschoot, P., Martinet, W., Maras,M., Neirynck, S., Contreras, R., Fiers, W., and Jou,W. M. (1999) Protection of mice against a lethal in-fluenza virus challenge after immunization withyeast-derived secreted influenza virus hemagglutinin.Eur. J. Biochem. 260, 166–175.

295. Martinet, W., Saelens, X., Deroo, T., Neirynck, S.,Contreras, R., Min Jou, W., and Fiers, W. (1997) Pro-tection of mice against a lethal influenza challengeby immunization with yeast-derived recombinant in-fluenza neuraminidase. Eur. J. Biochem. 247, 332–338.

296. Zhu, X., Wu, S., and Letchworth, G. J., III (1997)Yeast-secreted bovine herpesvirus type 1 glycopro-tein D has authentic conformational structure andimmunogenecity. Vaccine 15, 679–688.

297. Zhu, X., Wu, S., and Letchworth, G. J. (1999) A chi-meric protein comprised of bovine herpesvirus type 1glycoprotein D and bovine interleukin-6 is secretedby yeast and possesses biological activities of bothmolecules. Vaccine 17, 269–282.

298. Sugrue, R. J., Fu, J., Howe, J., and Chan, Y. C. (1997)Expression of the dengue virus structural proteins inPichia pastoris leads to the generation of virus-likeparticles. J. Gen. Virol. 78, 1861–1866.

299. Chiou, H. L., Lee, T. S., Kuo, J., Mau, Y. C., and Ho, M.S. (1997) Altered antigenicity of “a” determinant vari-ants of hepatitis B virus. J. Gen. Virol. 78, 2639–2645.

300. Eckart, M. R. and Bussineau, C. M. (1996) Qualityand authenticity of heterologous proteins synthesizedin yeast. Curr. Opin. Biotechnol. 7, 525–530.

301. Lal, S. K., Tulasiram, P., and Jameel, S. (1997) Ex-pression and characterization of the hepatitis E virusORF3 protein in the methylotrophic yeast, Pichiapastoris. Gene 190, 63–67.

302. Scorer, C. A., Buckholz, R. G., Clare, J. J., and Romanos,M. A. (1993) The intracellular production and secretion

52 Cregg et al.


of HIV-1 envelope protein in the meth-ylotrophic yeast Pichia pastoris . Gene 136,111–119.

303. Peng, Y. C. and Acheson, N. H. (1997) Produc-tion of active polyomavirus large T antigen inyeast Pichia pastoris. Virus Res. 49, 41–47.

304. Bisaillon, M., Bergeron, J., and Lemay, G. (1997)Characterization of the nucleoside triphosphate

phosphohydrolase and helicase activities of thereovirus lambda 1 protein. J. Biol. Chem. 272,18298–18303.

305. Bisaillon, M., Senechal, S., Bernier, L., and Lemay,G. (1999) A glycosyl hydrolase activity of mamma-lian reovirus sigma 1 protein can contribute to viralinfection through a mucus layer. J. Mol. Biol. 286,759–773.

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