2000 - James M. Cregg-Recombinant Protein Expression in Pichia Pastoris PDF

Recombinant Protein Expression
REVIEW
23
Recombinant Protein Expression in Pichia pastoris

James M. Cregg,1 Joan Lin Cereghino,2 Jianying Shi,3 and David R. Higgins*
Abstract
The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for
the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a largescale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program
to convert abundant methanol to a protein source for animal feed has been developed into what is today two
important biological tools: a model eukaryote used in cell biology research and a recombinant protein
production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial
availability of these tools coupled with a better understanding of the biology of Pichia species have led to
this microbes value and power in commercial and research labs alike.
Index Entries: Pichia pastoris; Pichia methanolica; methylotrophic yeast; heterologous protein production; foreign gene expression; yeast protein expression; fermentation.
1. Introduction
the strong preference of P. pastoris for respiratory growth, a key physiological trait that
greatly facilitates its culturing at high cell densities relative to fermentative yeasts; and
a decision in 1993 by Phillips Petroleum Company continued by Research Corporation Technologies (RCT) to release the P. pastoris
expression system to academic research laboratories, the consequence of which has been an
explosion in the knowledge base of the system.
The successful expression of more than 200 different heterologous proteins in P. pastoris has
been published (Fig. 1, Table 3). A web site has
been created and is maintained by the Cregg lab
that lists heterologous proteins expressed in
Pichia pastoris (http:// www.kgi.edu/html/
noncore/program4.htm#jc).
Pichia pastoris has become a highly successful system for the expression of heterologous
genes. Several factors have contributed to its
rapid acceptance, the most important of which
include:
a promoter derived from the alcohol oxidase I
gene (AOX1) of P. pastoris that is uniquely
suited for the controlled expression of foreign
genes;
the similarity of techniques needed for the
molecular genetic manipulation of P. pastoris
to those of Saccharomyces cerevisiae, one of
the best-characterized experimental systems in
modern biology;
*Author to whom all correspondence and reprint requests should be addressed: 1Keck Graduate Institute of Applied Life Sciences, 535 North
Watson Dr. Claremont, CA 91711, Email: James_Cregg@kgi.edu. 2Oregon Graduate Institute of Science and Technology, 20,000 N.W.
Walker Rd. Beaverton, OR 97006, E-mail: jlcereg@bmb.ogi.edu. 3 Oregon Graduate Institute of Science and Technology, 20,000 N.W.
Walker Rd. Beaverton, OR 97006, Email: jshi@bmb.ogi.edu. *Idun Pharmaceuticals,11085 N. Torrey Pines Road La Jolla, CA 92037, E
mail: dhiggins@idun.com.
Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 10736085/2000/16:1/2352/$17.50
MOLECULAR B IOTECHNOLOGY
23
Volume 16, 2000
24
Fig. 1. Results of Medline search conducted on

December 1, 1999, for the word Pichia in the title or
abstract. * = data for 1999 do not represent a complete
year.
As a yeast, P. pastoris is a single-celled

microorganism that is easy to manipulate and
culture. However, it is also a eukaryote and
capable of many of the posttranslational modifications performed by higher eukaryotic cells,
such as proteolytic processing, folding, disulfide bond formation, and glycosylation. Thus,
many proteins that end up as inactive inclusion
bodies in bacterial systems are produced as biologically active molecules in P. pastoris. The
P. pastoris system is also generally regarded as
being faster, easier, and less expensive to use
than expression systems derived from higher
eukaryotes, such as insect and mammalian tissue culture cell systems, and usually gives
higher expression levels.
A second role played by P. pastoris in
research is not directly related to its use as a
protein expression system. P. pastoris serves as
a useful model system to investigate certain
areas of modern cell biology, including the
molecular mechanisms involved in:
the import and assembly of peroxisomes;
the selective autophagic degradation of peroxisomes; and
the organization and function of the secretory pathway in eukaryotes.
In this review the basic aspects of the P.

pastoris expression system are highlighted.
Further information on the P. pastoris system
can be found in the numerous reviews describing the system (111). The DNA sequence of
MOLECULAR BIOTECHNOLOGY
Cregg et al.
many P. pastoris expression vectors and other
useful information can be found on the Invitrogen
web site (http://www.invitrogen.com).
2. A Brief History of the Pichia pastoris
Expression System
The ability of certain yeast species to utilize
methanol as a sole source of carbon and energy
was discovered approx 30 years ago by Koichi
Ogata (12). Because methanol could be inexpensively synthesized from natural gas (methane), there was immediate interest in exploiting
these organisms for the generation of yeast biomass or single-cell protein (SCP) to be marketed primarily as high-protein animal feed.
During the 1970s, Phillips Petroleum Company
(Bartlesville, OK) developed media and methods for growing P. pastoris on methanol in
continuous culture at high cell densities (>130
g/L dry cell weight) (13). However, during
this same period, the cost of methane
increased dramatically due to the oil crisis,
and the cost of soy beans (the major alternative source of animal feed protein) decreased.
As a result, the SCP process was never economically competitive.
In the early 1980s, Phillips Petroleum Company contracted with the Salk Institute Biotechnology/Industrial Associates, Inc. (SIBIA), a
biotechnology company located in La Jolla,
CA, to develop P. pastoris as a heterologous
gene expression system. Researchers at SIBIA
isolated the AOX1 gene (and its promoter) and
developed vectors, strains, and methods for
molecular genetic manipulation of P. pastoris
(1419). The combination of strong regulated
expression under control of the AOX1 promoter, along with the fermentation media and
methods developed for the SCP process,
resulted in strikingly high levels of foreign proteins in P. pastoris. In 1993, Phillips Petroleum
sold its patent position with the P. pastoris
expression system to RCT, the current patent
holder. In addition, Phillips Petroleum licensed
Invitrogen to sell components of the system to
researchers worldwide, an arrangement that
continues under RCT.
Volume 16, 2000

3. Pichia pastoris
as a Methylotrophic Yeast
P. pastoris is one of approx 30 yeast species
representing two different genera (Pichia and
Candida) capable of metabolizing methanol (20).
The Hansenula and Torulopsis genera are now
part of the Pichia and Candida genera,
repsectively (Hansenula polymorpha is now
Pichia angusta) (20). The methanol metabolic
pathway appears to be the same in all yeasts and
involves a unique set of pathway enzymes (21).
The first step in the metabolism of methanol is
the oxidation of methanol to formaldehyde, generating hydrogen peroxide in the process, by the
enzyme alcohol oxidase (AOX). To avoid hydrogen peroxide toxicity, this first step in methanol
metabolism takes place within a specialized
organelle, called the peroxisome, which sequesters toxic hydrogen peroxide away from the rest
of the cell. AOX is a homo-octomer with each
subunit containing one noncovalently bound FAD
(flavin adenine dinucleotide) cofactor. Alcohol
oxidase has a poor affinity for O2, and
methylotrophic yeasts appear to compensate for
this deficiency by synthesizing large amounts of
the enzyme.
There are two genes in P. pastoris that code for
AOXAOX1 and AOX2but the AOX1 gene is
responsible for the vast majority of alcohol oxidase activity in the cell (18). Expression of the
AOX1 gene is tightly regulated and induced by
methanol to high levels. In methanol-grown
shake-flask cultures, this level is typically approx
5% of total soluble protein but can be 30% in
cells fed methanol at growth limiting rates in fermentor cultures (22). Expression of the AOX1
gene is controlled at the level of transcription
(12,16,18). In methanol-grown cells, approx 5%
of polyA+ RNA is from the AOX1 gene, whereas,
in cells grown on other carbon sources, the AOX1
message is undetectable. The regulation of the
AOX1 gene is similar to the regulation of the
GAL1 gene of S. cerevisiae, in that control
appears to involve two mechanisms: a repression/
derepression mechanism plus an induction
mechanism. However, unlike GAL1 regulation,
25
derepressing conditions (e.g., the absence of a
repressing carbon source such as glucose in the
medium) do not result in substantial transcription
of the AOX1 gene. The presence of methanol
appears to be essential to induce high levels of
transcription (16).
4. Secretion of Heterologous Proteins

With P. pastoris, heterologous proteins can
either be expressed intracellularly or secreted into
the medium. Because P. pastoris secretes only
low levels of endogenous proteins and because its
culture medium contains no added proteins, a
secreted heterologous protein comprises the vast
majority of the total protein in the medium
(23,24). Thus, secretion serves as a major first
step in purification, separating the foreign protein
from the bulk of cellular proteins. However, the
option of secretion is usually limited to foreign
proteins that are normally secreted by their native
hosts. Secretion requires the presence of a signal
sequence on the foreign protein to target it to the
secretory pathway. Although several different
secretion signal sequences have been used successfully, including the native secretion signal
present on some heterologous proteins, success
has been variable. The secretion signal sequence
from the S. cerevisiae -factor prepro peptide has
been used with the most success.
5. Common Expression Strains
All P. pastoris expression strains are derivatives of NRRL-Y 11430 (Northern Regional Research Laboratories, Peoria, IL) (Table 1). Most
have a mutation in the histidinol dehydrogenase
gene (HIS4) to allow for selection of expression
vectors containing HIS4 upon transformation
(10,11,15). Other biosynthetic gene/auxotrophic
mutant host marker combinations are also available but are used less frequently. All of these
strains grow on complex media but require
supplementation with histidine (or other appropriate nutrient) for growth on minimal media.
Three types of host strains are available that
vary with regard to their ability to utilize methanol due to deletions in one or both AOX genes.
Strains with deleted AOX genes sometimes are
Volume 16, 2000
26
Cregg et al.
Table 1
P. pastoris Expression Host Strains
Strain
Name
Y-11430
GS115
KM71
MC100-3
SMD1168
SMD1165
SMD1163
Genotype
his4
aox1::SARG4 his4 arg4
aox1::SARG4 aox2::Phis4
his4 arg4
pep4 his4
prb1 his4
pep4 prb1 his4
Phenotype
Wild type
Mut+ His
Muts His
Mut His
Mut+ His, protease-deficient
Reference
NRRL*
15
16
18
10,11
10,11
10,11
*Northern Regional Research Laboratories, Peoria, IL.
better producers of a foreign protein than wildtype strains (21,23,24). These strains also require
much less methanol to induce expression, which
can be useful in large fermentor cultures where a
large amount of methanol is sometimes considered a significant fire hazard. However, the most
commonly used expression host is GS115 (his4),
which is wild-type with regard to the AOX1 and
AOX2 genes and grows on methanol at the wildtype rate (methanol utilization plus [Mut+] phenotype). KM71 (his4 arg4 aox1::ARG4) is a
strain in which the chromosomal AOX1 gene is
largely deleted and replaced with the S. cerevisiae
ARG4 gene (17). As a result, this strain must rely
on the much weaker AOX2 gene for AOX and
grows on methanol at a slow rate [methanol utilization slow (Muts) phenotype]. With many P.
pastoris expression vectors, it is possible to insert
an expression cassette and simultaneously delete
the AOX1 gene of a Mut+ strain (10,11,25). The
third host MC100-3 (his4 arg4 aox1::SARG4
aox2::Phis4) is deleted for both AOX genes and
is totally unable to grow on methanol (methanol
utilization minus [Mut] phenotype) (10,11,28,26).
Some secreted foreign proteins are unstable in
the P. pastoris culture medium in which they are
rapidly degraded by proteases. Major vacuolar
proteases appear to be a significant factor in degradation, particularly in fermentor cultures, owing
to the high cell density environment in combination with the lysis of a small percentage of cells.
The use of host strains that are defective in these
proteases has proven to help reduce degradation
in several instances (10,11). SMD1163 (his4 pep4

prb1), SMD1165 (his4 prb1), and SMD1168
(his4 pep4) are protease-deficient strains that may
provide a more suitable environment for expression of certain heterologous proteins. The PEP4
gene encodes proteinase A, a vacuolar aspartyl
protease required for the activation of other vacuolar proteases, such as carboxypeptidase Y and
proteinase B. Proteinase B, prior to processing
and activation by proteinase A, has about half the
activity of the processed enzyme. The PRB1 gene
codes for proteinase B. Therefore, pep4 mutants
display a substantial decrease or elimination in
proteinase A and carboxypeptidase Y activities,
and partial reduction in proteinase B activity. In
the prb1 mutant, only proteinase B activity is
eliminated, whereas pep4 prb1 double mutants
show a substantial reduction or elimination in all
three of these protease activities.
6. Expression Vectors
Plasmid vectors designed for heterologous protein expression in P. pastoris have several common features (Table 2). The foreign gene
expression cassette is one of those and is composed
of DNA sequences containing the P. pastoris
AOX1 promoter, followed by one or more unique
restriction sites for insertion of the foreign gene,
followed by the transcriptional termination
sequence from the P. pastoris AOX1 gene that
directs efficient 3' processing and polyadenylation
of the mRNAs. Many of these vectors also include
the P. pastoris HIS4 gene as a selectable marker
Volume 16, 2000
Table 2
Common P. pastoris Expression Vectors
Selectable
markers
Features

Vector
name
Targeting
pHIL-D2
Intracellular
HIS4
pAO815
Intracellular
HIS4
pPIC3K
Intracellular
HIS4 and kanr
pPICZ
Intracellular
bler
pHWO10
Intracellular
HIS4
pGAPZ
Intracellular
bler
pHIL-S1
Secreted
HIS4
pPIC9K
Secreted
HIS4 and kanr
pPICZ
Secreted
bler
pGAPZ
Secreted
bler
NotI sites for AOX1 gene

replacment
Expression cassette sites
bounded by BamHI and BglII
for generation of multicopy
expression vector
Multiple cloning sites;
for insertion of foreign genes;
G418 selection for multicopy strains
Multiple cloning sites for
insertion of foreign genes;
Zeocin selection for multicopy
strains; potential for fusion of
foreign protein to His6
and myc epitope tags
Expression controlled
by constitutive GAPp
Expression controlled
by constitutive GAPp; multiple
cloning site for insertion of
foreign genes; Zeocin selection
for multicopy strains; potential
for fusion of foreign protein
to His6 and myc epitope tags
AOX1p fused to PHO1
secretion signal; XhoI, EcoRI,
and BamHI sites available for
insertion of foreign genes
AOX1p fused to -MF prepro
signal sequence; XhoI (not
unique), EcoRI, NotI, SnaBI,
and AvrII sites available for
insertion of foreign genes;
G418 selection for multicopy
strains
AOX1p fused to -MF prepro
signal sequence; multiple
cloning site for insertion
of foreign genes; Zeocin
selection for multicopy strains;
potential for fusion of foreign
protein to His6 and myc
epitope tags
Expression controlled by
constitutive GAPp; GAPp
fused to -MF prepro signal
sequence; multiple cloning site
for insertion of foreign genes;
Zeocin selection for multicopy
strains; potential for fusion of
foreign protein to His6 and
myc epitope tags
27
References
Sreekrishna,
personal comm.
2
10
29
Invitrogen,
Carlsbad, CA
Sreekrishna,
personal comm;
Invitrogen,
Carlsbad, CA
32
10
Invitrogen,
Carlsbad, CA
Volume 16, 2000
28
for transformation into his4 mutant hosts of P.
pastoris, as well as sequences required for plasmid
replication and maintenance in bacteria (i.e., ColE1
replication origin and ampicillin-resistance gene).
Some vectors also contain AOX1 3' flanking
sequences that are derived from a region of the P.
pastoris genome that lies immediately 3' of the
AOX1 gene and can be used to direct fragments
containing a foreign gene expression cassette to
integration at the AOX1 locus by gene replacement
or gene insertion 3' to AOX1 gene (10,11).
Additional features that are present in certain
P. pastoris expression 2vectors serve as tools for
specialized functions. For secretion of foreign
proteins, vectors have been constructed that contain a DNA sequence immediately following the
AOX1 promoter that encodes a secretion signal.
The most frequently used of these is the S.
cerevisiae -factor prepro signal sequence
(10,11,27,28). However, vectors containing the
signal sequence derived from the P. pastoris acid
phosphatase gene (PHO1) are also available.
Vectors with dominant drug-resistance markers that allow for enrichment of strains that
receive multiple copies of foreign gene expression cassettes during transformations have been
developed. One set of vectors (pPIC3K and
pPIC9K) contains the bacterial kanamycin-resistance gene and confers resistance to high levels
of G418 upon strains that contain multiple copies of these vectors (10,11,28). Another set of
vectors (the pPICZ series) contains the Sh ble
gene from Streptoalloteichus hindustanus
(10,11). This gene is small (375 bp) and confers
resistance to the drug Zeocin in E. coli, yeasts
(including P. pastoris), and other eukaryotes.
Because the ble gene serves as the selectable
marker for both E. coli and P. pastoris, the
ZeoR vectors are much smaller (approx 3 kb)
and easier to manipulate than other P. pastoris
expression vectors. These vectors also contain
a multiple cloning site (MCS) with several
unique restriction sites for convenience of foreign gene insertion and sequences encoding the
His6 and myc epitopes so that foreign proteins
can be easily epitope-tagged at their carboxyl
termini, if desired.
Cregg et al.
Another feature present on certain vectors (e.g.,
pAO815 and the pPICZ vector series) is designed
to facilitate the construction of expression vectors
with multiple expression cassette copies (10).
Multiple copies of an expression cassette are
introduced in these vectors by inserting an expression cassette bounded by a BamHI and a BglII site
into the BamHI site of a vector already containing
a single expression cassette copy. The resulting
BamHI/BglII junction between the two cassettes
can no longer be cleaved by either enzyme allowing for the insertion of another BamHIBglIIbounded cassette into the same vector to generate
a vector with three cassette copies. The process of
addition is repeated until 68 copies of a cassette
are present in a single final vector, which is then
transformed into the P. pastoris host strain.
Recently, vectors containing alternative promoters have become available (Table 2). Unlike
the AOX1 promoter, these promoters do not
require induction by methanol, which may be
problematic in some instances. One is a strong
constitutive promoter derived from the P. pastoris
glyceraldehyde-3-phosphate dehydrogenase gene
(GAP) (29). In addition to not involving methanol, the GAP promoter is convenient since cultures do not need to be shifted from one carbon
source to another to induce expression of a foreign gene. However, this promoter is not suitable
for the expression foreign genes whose products
are toxic to P. pastoris since the constant highlevel expression can result in the selection of
nonexpressing derivative strains that have either
lost the expression vector or have gained mutations that reduce or eliminate expression of the
foreign gene.
The second promoter is from the P. pastoris
formaldehyde dehydrogenase gene (FLD1) (30).
FLD1 is required for growth of P. pastoris on either methanol as a sole carbon source or certain
methylated amines such as methylamine as a sole
nitrogen source and is strongly induced by either
of these conditions. As a result, expression of a
foreign gene placed under control of the FLD1
promoter is repressed in media containing glucose
and ammonium ions but can be independently
induced using media containing either methanol
Volume 16, 2000

or methylamine. With the FLD1 promoter, only
one expression vector and strain need be constructed to examine expression under both these
conditions. Importantly, both the GAP and FLD1
promoters express foreign genes at levels comparable to those observed with the AOX1 promoter.
7. Integration of Vectors
into the Pichia pastoris Genome
As in S. cerevisiae, linear vector DNAs can
generate stable transformants of P. pastoris via
homologous recombination between sequences
shared by the vector and host genome
(10,11,15,25). Such integrants show strong stability in the absence of selective pressure even when
present as multiple copies. All P. pastoris expression vectors carry at least one P. pastoris DNA
segment (the promoter fragment) with unique
restriction sites that can be cleaved and used to
direct the vector to integrate into the host genome
by a single crossover type insertion event. Vectors containing the P. pastoris HIS4 gene can also
be directed to integrate into the P. pastoris
genomic his4 locus.
Expression vectors that contain 3' AOX1
sequences can be integrated into the P. pastoris
genome by a single crossover event at either
AOX1 or HIS4 loci or by a gene replacement (
insertion) event at AOX1. The latter event arises
from crossovers at both the AOX1 promoter and
3' AOX1 regions of the vector and genome, and
results in the deletion of the AOX1 coding region
(i.e., gene replacement). Transformants resulting
from such an AOX1 replacement event are phenotypically His+ and Muts. Muts strains sometimes
express higher levels of foreign protein (10). In
addition, a Muts phenotype serves as a convenient
indicator to confirm the presence of an integrated
expression cassette in the P. pastoris genome.
With either single crossover or gene replacement integration strategies and selection for His+
transformants, a significant percentage of
transformants will not contain the expression vector. This appears to be due to gene conversion
events between the HIS4 gene on the vector and
the P. pastoris his4 locus such that the wild-type
HIS4 gene recombines into the genome without
29
any additional vector sequences. These events
account for 1050% of His+ transformant colonies and appear to occur at highest frequency
when using electroporation to introduce vector
DNAs.
Multiple gene insertion events at a single locus
occur spontaneously at a low but detectable frequencybetween 1 and 10% of His+ transformants (10,11,31,32). Multicopy events can occur
as gene insertions either at the AOX1 or his4 loci
and can be detected by DNA analysis methods
(e.g., PCR, Southern/dot blotting, or differential
hybridization) (3234) or by methods that directly
examine levels of the foreign protein (e.g., activity
assay, SDS-PAGE, or colony immunoblotting)
(31,32,35). It is possible to enrich transformant
populations for ones that have multiple copies of
an expression vector by use of either a G418R
or ZeoR gene-containing vector and selecting
for hyper-resistance to the appropriate drug
(10,11,28,32). It is important to note that, with the
G418R vectors, it is essential to first select for
His+ transformants and to then screen for ones that
are resistant to G418R (32). With ZeoR vectors, it
is possible to directly select for hyper-Zeo-resistant transformants (10). Drug-resistant strains resulting from either the G418R or ZeoR selection
methods can contain between one and five copies
of the expression vector. To find strains with 20
or more copies, it is usually necessary to screen
more than 100 drug-resistant strains.
8. Posttranslational Modifications
P. pastoris has the potential to perform many
of the posttranslational modifications typically
associated with higher eukaryotes. These include
processing of signal sequences (both pre- and
prepro-type), folding, disulfide bridge formation
(10,11,36), and O- and N-linked glycosylation.
Glycosylation of secreted foreign (higher)
eukaryotic proteins by P. pastoris and other fungi
can be problematic. In mammals, O-linked oligosaccharides are composed of a variety of sugars including N-acetylgalactosamine, galactose,
and sialic acid. In contrast, lower eukaryotes,
including P. pastoris, add O-oligosaccharides
solely composed of mannose (Man) residues
Volume 16, 2000
30
(4,10,11). The number of Man residues per chain,
their manner of linkage, and the frequency and
specificity of O-glycosylation in P. pastoris have
yet to be determined. One should not assume that,
because a protein is not O-glycosylated by its
native host, P. pastoris will not glycosylate it. P.
pastoris added O-linked mannose to approx 15%
of human IGF-1 protein, although this protein is
not glycosylated at all in humans. Furthermore,
one should not assume that the specific Ser and
Thr residue(s) selected for O-glycosylation by P.
pastoris will be the same as the native host.
N-glycosylation in P. pastoris and other fungi is
also different than in higher eukaryotes (4,10,11). In
all eukaryotes, it begins in the endoplasmic reticulum
with the transfer of a lipid-linked oligosaccharide unit
(Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = Nacetylglucosamine) to asparagine at the recognition
sequence Asn-X-Ser/Thr. This oligosaccharide core
unit is subsequently trimmed to Man8GlcNAc2. It is
at this point that lower and higher eukaryotic
glycosylation patterns begin to differ. The mammalian Golgi apparatus performs a series of trimming and addition reactions that generates
oligosaccharides composed of either Man5
6GlcNAc2 (high-mannose type), a mixture of several different sugars (complex type), or a
combination of both (hybrid type). Two distinct
patterns of N-glycosylation have been observed
on foreign proteins secreted by P. pastoris. Some
proteins, such as S. cerevisiae invertase, are secreted with carbohydrate structures similar in size
and structure to the core unit (Man811GlcNAc2)
(26,36).
Other foreign proteins secreted from P.
pastoris receive much more carbohydrate and appear by SDS-PAGE and Western blotting to be
hyperglycosylated (10,11,23). Interestingly, P.
pastoris does not appear to be capable of adding
1,3-terminal mannose to oligosaccharides (R.
Trimble, personal communication). This contrasts
with S. cerevisiae oligosaccharides where 1,3linked terminal mannose is common. Aside from
the probable absence of 1,3-linked mannose, little
is known regarding the structure of P. pastoris
outer-chain oligosaccharides. Furthermore, it is
also not clear why outer chains are added to some
Cregg et al.
P. pastoris-secreted proteins and not others, nor
how outer chain addition may be prevented.
N-linked high-mannose oligosaccharides
added to proteins by yeasts represent a significant
problem in the use of foreign-secreted proteins by
the pharmaceutical industry. They can be exceedingly antigenic when introduced intravenously
into mammals and are rapidly cleared from the
blood by the liver. An additional problem caused
by the differences between yeast and mammalian
N-linked glycosylation patterns is that the long
outer chains can potentially interfere with the
folding or function of a foreign protein.
9. Expression in Fermentor Cultures

Although a few foreign proteins have expressed
well in P. pastoris shake-flask cultures, expression
levels in shake-flasks are typically low relative to
what is obtainable in fermenter cultures. One reason fermenter culturing is necessary is that only in
the controlled environment of a fermenter is it possible to grow the organism to high cell densities
(>100 g/L dry cell weight or 500 OD600 units/mL).
Especially for secreted proteins, the concentration
of product in the medium is roughly proportional
to the concentration of cells in culture. A second
reason is that the level of transcription initiated
from the AOX1 promoter can be 35 times greater
in P. pastoris cells fed methanol at growth-limiting rates in fermenter culture relative to cells grown
in excess methanol. Thus, even for intracellularly
expressed proteins, yields of product from a given
strain as a percentage of total cellular proteins are
significantly higher from fermenter cultured cells.
A third reason is that methanol metabolism utilizes
oxygen at a high rate, and expression of foreign
genes is negatively affected by oxygen limitation.
Only in the controlled environment of a fermenter
is it feasible to accurately monitor and adjust oxygen levels in the culture medium. Thus, most users
of the P. pastoris expression system should expect
to produce their foreign protein in fermenters.
A hallmark of the P. pastoris system is the ease
by which expression strains scale up from shakeflask to high-density fermenter cultures. Considerable effort has gone into the optimization of
high-cell-density fermentation techniques for
Volume 16, 2000
31
Table 3
Heterologous Proteins Expressed in Pichia.pastoris
Protein
Comments
Mode, amount, signal sequence
References
Bacteria
Bacillus licheniformis -amylase
Bacillus stearothermophilus
D -alanine carboxypeptidase
Bordetella pertussis pertussis pertactin (P69)
Clostridium botulinum neurotoxin
(BoNT) serotype A and B
heavy chain fragment, serotype B
serotype A binding domain
Clostridium tetani tetanus toxin fragment C
Escherichia coli acid
phosphatase/phytase (appA2)
Escherichia coli -galactosidase
Escherichia coli -lactamase
Leishmania major cathepsin B-like protease
Staphylococcus aureus staphylokinase
Streptococcus equisimili streptokinase
Streptomyces subtilisin inhibitor
Streptomyces viridosporus T7A
peroxidase, endoglucanase
Toxoplasma gondii SAG1 antigen
Vibrio cholerae accessory
cholera enterotoxin (Acc)
S, 2.5 g/L, SUC2

S, 100 mg/L, native
53, 54
55
I, 3 g/L
I, 78 mg/L
34
56
I, 390 g/g
57
I, 2.4 mg total
58
I, 12 g/L
S, 28.9 U/mg
33
59
I, 2.0 103 U/mg

I
S, -MF
S, 50 mg/L, -MF
I, 77 mg/L
S
S, 2.47 g/L total protein, -MF
16
29
60
61
62
63
64
S, 12 mg/L, -MF
S, 7 mg/L, -MF
65
66
S, -MF
S, 400 mg/L, native
S, 400 mg/L, PHO1
67
68
69
S, 2.3 g/L, PHO1

S, PHO1
70
71
S, 0.15 mg/L, PHO1
72
S, 1 mg/L, synthetic native, PHO1

S, 65 U/ml, -MF
I, 0.65 U/mg; S, 0.18 U/mg, -MF
S, 150 U/ml, -MF
S, 1 mg/L, PHO1
S, native
S, 60 mg/L, -MF
S, 45 mg/L, PHO1
S, 60 mg/L, -MF
S, 2.5 g/L, native
S, 400 mg/L, PHO1
S, 40 mg/L, PHO1
73
74
75
76
77
78
79
80
81
23
82
82
Fungi
Alternaria Alt 1 allergen
Aspergillus awamori glucoamylase
Aspergillus awamori glucoamylase
catalytic domain
Aspergillus fumigatus catalase L
Aspergillus fumigatus dipeptidyl
peptidase IV (DPP IV)
Aspergillus fumigatus dipeptidyl
peptidase V (DPP V)
Aspergillus giganteus -sarcin ribotoxin
Aspergillus niger phytase (phyA)
Candida guilliermondii xylose reductase gene (xylI)
Candida rugosa lipase 1 (CRL)
Fusarium solani pectate lyase (pelC)
Fusarium solani pectate lyase (pelD)
Geotrichum candidum lipases isoenzymes
Phytophthora cryptogea -cyptogein
Rhizopus oryzae lipase
Saccharomyces cerevisiae invertase
Saccharomyces cerevisiae Ktr1p
Saccharomyces cerevisiae
Volume 16, 2000
32
(-1,2-mannosyltransferase)
Schizophyllum commune vitamin B2aldehyde-forming enzyme
Trametes versicolor (white rot fungus)
laccase (lccI)
Trichoderma harzianum -(1-6)-glucanase
Cregg et al.
S, 120 mg/L, -MF
83
S, native and -MF
84
S, 9.3 mg/L
85
I
I
86
87
S, 24 mg/L, -MF
40
S, 50 mg/L, PHO1
88
I, 400 g/g
89
S, 11.5 g/L, native
90
I, 2.167 U/g
I, 18 g/g
91
92, 93
S, -MF
94
S, 50 mg/L, native
S, 1 mg/L, native
S, -MF
S, 400 mg/L, -MF
S, 1 mg/L, native
S, 1.5 g/L, PHO1
S, PHA-E
I, 22 g/L
S, 10 mg/L, -MF
S, native
I, 30 g/g
I, 20 g/g
S, 60 mg/L, -MF
S, -MF
S, native
S, 50 mg/L, -MF
95
95
96
97
98
99, 100
101
102
103, 104
105
106, 107
108
109
110
110
111
S, native
101
S, -MF
I, 25 g/g
S, 1 mg/L, -MF
S, 10 mg/L, -MF
I, 250 U/g
I, 0.5 mg/g
S, PHO1
S, -MF
S, PHO1
112
113
114
115
116, 117
118
119
120
121
S, 720 mg/L, PHO1
122
Protists
Chondrus crispus red alga hexose oxidase
Gracilariopsis lemaneiformis red alga
-1,4-glucan lyase (GLq1)
Plasmodium falciparum merozoite
surface protein 1 (MSP-1)
Plasmodium vivax apical
Membrane antigen I (AMA-1)
Reticulomyxa filosa (giant freshwater amoeba)
2, 2 tubulin isoforms
Trypanosoma cruzi acid -mannosidase
Plants
Allium sativum (garlic) alliin lyase
Arabidopsis thaliana
NADH:nitrate reductase
Barley (Hordeum vulgare) sucrose
fructan 6-fructosyl transferase
Barley -amylase 1
Barley -amylase 2
Barley aleurone tissue -glucosidase
Coffee bean -galactosidase
Cynara cardunculus (cardoon) cyprosin
Cynodon dactylon (Bermuda grass) Cyn d 1
Galanthus nivalis agglutinin
Hevea brasiliensis hydroxynitrile lyase
Hevea brasiliensis Hev b 7 patatin-like allergen
Maize cytokinin oxidase
Oat phytochrome A, phA
Oat phytochrome A, phyA65 apoprotein
Olea europaea (olive tree) aeroallergen Ole e 1
Pepper endo--1,4-glucanase cCel1
Pepper endo--1,4-glucanase cCel2
Persea americana (avocado)
prs a 1 major allergen
Phaseolus vulgaris agglutinin
(phytohaemagglutinin)
Phleum pratense -expansin
Potato phytochrome B
Ragweed allergen Amb a 6
Soybean root nodule acid phosphatase
Spinach glycolate oxidase
Spinach phosphoribulokinase
Sugar beet defensin AX2
Timothy grass group I allergen
Tomato Lycopesicon esculentum Mill.
LeMir (L. esculentum miraculin)
Wheat lipid transfer protein
Volume 16, 2000
33
Invertebrates
Achacina fulica Ferussac
(giant African snail) achacin
Aplysia californica (marine invertebrate)
ADP ribosyl cyclase
Aqueora victoria (jellyfish)
green fluorescent protein
Boophilus microplus (cattle tick) Bm86
Cockroach allergen, Bla g 4
Drosophila melanogaster
angiotensin I-converting enzyme
Drosophila melanogaster
caritine palmitoyltransferase
Firefly luciferase
GAVAC vaccine against cattle tick
Haementeria ghilanii
(South American leech) ghilanten
Hirudo medicinalis (leech) hirudin
Honeybee odorant-binding protein (ASP2)
Honeybee olfactory protein
Nippostrongylus brasiliensis
(parasitic nematode) nonneuronal secreted
acetylcholine sterase
Spider dragline silk protein
Tick anticoagulant peptide
Yellowjacket venom allergen, Ag5
S, 0.2 mg/L, native
123
S, 300 mg/L, -MF
124
I, S, PHA-E
101,125
I, S*, 1.5 g/L, SUC2

S, 50 mg/L
S, 160 mg/L, -MF
126129
130
131
I (mitochondria)
132
I (peroxisome)
S, 2.0 g/L
S, 10 mg/L, -MF
133
134
135
S, 1.5 g/L, -MF

S, 150 mg/L, native
S, 0.2 g/L, native
136
137
138
139
S, 27 mg/L, -MF
I, 663 mg/L
S, 1.7 g/L
S, 100 mg/L, -MF
140
27
141
S, 6.3 mg/L, -MF

S, 4 g/ml, -MF
S, 4 mg/L, SUC2
S, 550 mg/L, native
S*, 0.3 mg/L, PHO1
S, 930 mg/L, -MF
I, 1 g/L
S, >1 g/L, -MF
S, 1.1 mg/L, -MF
S, -MF
S, -MF
S, 2 mg/L, native
142
143
144
145
146
147
148
4244
149
150
151
152
S, 11.6 mg/L, -MF, PHO1

S, native
153
154
S, 20 mg/L, -MF
S, mg amounts, native
S*, 40 pmol/mg, -MF
S, 450 mg/L, -MF
S, 14.8 g/l, -MF
S, 10 mg/L, -MF
S, native
S, 270 mg/L, native
I (membrane-bound), 6 g/mg
S, 250 mg/L, a-MF, PHO1
45
155
156
28
157
158
159
160
161163
164
Vertebrates (nonhuman)
Bovine enterokinase catalytic domain
Bovine follicle-stimulating hormone -subunit
Bovine IFN- 1
Bovine lysozyme c2
Bovine opsin
Bovine pancreatic trypsin inhibitor (aprotinin)
Bovine -casein
Bovine -lactoglobulin
Bovine tissue-type plasminogen activator (tPA)
Bovine transcobalamin
Brushtail possum TNF
Bungarus fasciatus (snake) venom
gland acetylcholinesterase
Chicken liver -N-acetylgalactosaminidase
Electrophorus electricus
acetylcholinesterase AChE type T
Hen lysozyme
Mammalian lipocalin allergen Bos d2
Mouse 5HT5A 5-tryptamine receptor
Mouse epidermal growth factor
Mouse gelatin
Mouse gelatinase B
Mouse lysosomal acid -mannosidase
Mouse major urinary protein complex (MUP)
Mouse Mdr3 P-glycoprotein
Mouse single-chain Fv fragments (sFv)
Volume 16, 2000
34
Murine endostatin
Murine Golgi mannosidase IA
Murine macrophage
inflammatory protein-2 (MIP-2)
Ovine follicle-stimulating hormone (oFSH)
Porcine carboxypeptidase B
Porcine follicle-stimulating hormone
Porcine inhibitor of carbonic
anhydrase (transferrin family)
Porcine leukocyte 12-lipoxygenase
Rabbit intestinal peptide transporter (PEPT1)
Rabbit intestinal peptide transporter (PEPT2)
Rabbit monoclonal single-chain Fv specific for
recombinant human leukemia inhibitory factor
Rabbit plasma cholesteryl ester transfer protein
Rabbit testicular angiotensin-converting enzyme
Rat acetycholinesterase
Rat brain acetylcholinesterase T subunit
Rat complement regulator, crry
Rat Golgi sialoglycoprotein MG160
Rat high-mobility group 1 (HMG 1)
Rat liver mitochondrial carnitine palmitoyl
transferases I and II (CPTI and II)
Rat neural cell adhesion molecule
Rat NO synthase reductase domain
Rat peroxisomal multifunctional
enzyme (perMFE-II)
Rat procathepsin B
Sea raven type II antifreeze protein (SRAFP)
Shark 17-hydroxylase/C17,20-lyase
Syrian golden hamster prion protein PrPc
Cregg et al.
S, 200 mg/L, -MF
S, PHO1
S, 40 mg/L, -MF
165
166
167
S, 22 mg/L, -MF
S, 200 mg/L, -MF
S, 10 mg/L, PHO1
S, 5 mg/L, -MF
168
169
170
171
I
I
I
S, 100 mg/L, -MF
172
173
174
175
S, PHO1
S, PHO1, native
S, 1 mg/L, native
S, 100 U/L -MF
S, -MF
S, 10 mg/L, -MF
S, 50 mg/L, -MF
I (mitochondria)
176
177
152
178
179
180
181
182,183
S, -MF
I, 25 mg/L
I
184
185
186
S, 100 mg/L, -MF

S, 30 mg/L, -MF
I
I, <0.1 mg/L
187,188
37,189
190
191
S, 30 mg/L, -MF
S, -MF
S, 11.6 mg/L, -MF
S, inulinase signal sequence
I
S*, 25 nmol/g, -MF
S*, a-MF
I, 1 mg/L
126
192
193
194
195
156
196
197
S, PHO1
198
S, 24 mg/L, 0.1 mg/L, -MF
199
S, 40 mg/L, -MF
S, 24 mg/L, PHO1
S, 4.5 + 1 mg/L, native
S, 300 mg/L, native, INV
S, 1 mg/L, -MF
200
201203
204
205
206
I, 0.2 mg/L
S, 20 mg/L, -MF
207,208
209
Humans
(1,3/4) Fucosyltransferase
-1,2-Mannosidase 1B w/o TM domain
-N-Acetylgalactosaminidase (-NAGAL)
1-Antitrypsin (1-AT)
2-Antiplasmin
2-Adrenergic receptor
-Opioid receptor
ADAR1, ADAR2, ds-RNA-specific
adenosine deaminases
Alzheimers disease amyloid precursor protein
, , and -secretase products
Alzheimers disease amyloid
precursor protein, 2 domains
Amyloid precursor-like protein 2 (APLP2)
Amyloid precursor protein (APP)
Amyloid precursor proteins, rAPP695, rAPP770
Bile salt-stimulated lipase
Bivalent diabody against carcinoembryonic
antigen (CEA), T-cell coreceptor CD2
c-Kit receptor kinase domain
Carcinoembryonic antigen
Volume 16, 2000

Caspase-3
Cathepsin K
Cathepsin L propeptide
Cathepsin V
Cathepsin X
CD38
CD40 ligand soluble form
Chimeric B7-2 antibody fusion protein
Chorionic gonadotropin
subunit, subunit, and heterodimer
Cromer blood group antigen
decay-accelerating factor
Cytomegalovirus ppUL44 antigen
Decay-accelerating factor DAF
(CD55)-Echovirus-7-receptor
Double-stranded RNA-specific editase I (hREDI)
Endostatin
Erythropoietin (EPO) receptor
Fas ligand
Fibrinogen, 143411, 143427
Fibroblast collagenase (proMMP-1)
Fibrinogen-420 EC domain
Gastric cathepsin E
Gelatinase B
Granzyme B
Heart muscle carnitine palmitoyltransferase I (M-CPTI)
Insulin
Insulin-like growth factor-1 (IGF-1)
Interferon- receptor cytoplasmic domain
15N-Interferon
Interleukin-17 (hIL-17)
Intracellular proteinase inhibitor (PI-6)
Kunitz-type protease inhibitor domain
of protease nexin-2/amyloid -protein precursor
Leukemia inhibitory factor (LIF)
Lymphocyte surface antigen CD38
Lysosomal -mannosidase
Mast cell tryptase
MHC class II heterodimers
(soluble form/HLA-DR2)
Monoclonal single-chain Fv
Monocyte chemoattractant protein-1 (MCP-1)
Monocyte chemotactic protein-3 (hMCP-3)
NEFA (DNA binding, EF-hand,
Acidic amino acid rich region)
Neural cell adhesion molecule (NCAM)
NonO nucleic acid binding protein
Oncostatin-M
Pancreatic -amylase
Pancreatic triglyceride lipase
Papain nitrile hydratase
Placental alkaline phosphatase (PLAP)
35
I, 1 g/g
S, 38 mg/L, -MF
S, 10 mg/L, -MF
S, -MF
S, 5 mg/L, -MF
S, 455 mg/L, -MF
S, 255 mg/L
S, 15 mg/L, -MF
S, 24 mg/L (), 3mg/L (),
16 mg/L (), -MF
S, -MF
221
I, 0.1 mg/mL
S, 6 mg/L, -MF
222
223
I, 1 mg/L
S, 20 mg/L, -MF
S, 200 mg/L, PHO1
S, 100 mg/L, -MF
S, 100 mg/L, 75 mg/L, -MF
S, 2.3 mg/L, -MF
S, -MF
S, 0.6 mg/L, native
S, 50 mg/L, -MF
S, 1 mg/L, -MF
I (mitochondria)
224
165,225
226
227
228
229
230
231
232
233
234
S, synthetic signal
S, 600 mg/L, -MF
I
S, 810 mg/L, PHO1
S, 0.35 mg/L, -MF
I, 50 mg/L
S, 1.0 g/L, -MF
235
236
237
238
239
240
S, 17 mg/L, -MF
S, 400 mg/L, PHO1
S, 83 g/L, native
S, 6.5 mg/L, -MF
S, 400 g/L, -MF
210
211,212
213,214
215
216
217
218
219
220
241
242
243
244
245,246
S, 50 mg/L, -MF
S, 100 mg/L, native or -MF
S, 1 mg/L, PHO1
S, 1001200 mg/L, -MF
247
248
249
250
251
S, 50 mg/L, PHO1
I (endoplasmic reticulum)
S, 50 mg/L, -MF
S, 20 mg/L, -MF
S, 75 ml/L, PHO1
S, 5 mg/L, -MF
S, 2 mg/L, PHO1
252
253
254
255
256
257
258
Volume 16, 2000
36
Cregg et al.
Placental protein-14 (PP-14)

Plasminogen kringles 1-4
Plasminogen kringles 1-4, angiostatin protein
Plasminogen kringles K2
Procarboxypeptidase A2
Procathepsin B
Procolipase
Protein kinase C interacting protein-1 (PKCI-1)
Proteinase 3, Wegeners antigen
Proteinase inhibitor 8
scFv (against ovarian carcinoma)biotin mimetic peptide
scFv (against squamous carcinoma)
Serum albumin
Serum transferrin N-Lobe
Sex steroid binding protein
Single-chain urokinase-type
plasminogen activator
Thrombomodulin
Tissue factor extracellular domain
Tissue kallikrein
Tissue-type plasminogen activator
kringle 2 domain
Transforming growth factor
receptor extracellular domain
Tumor necrosis factor (TNF)
Type 1 plasminogen activator inhibitor (PAI-1)
Type III collagen (with prolyl 4-hydroxylase)
Urokinase-type plasminogen activator-annexin V
chimera
S, -MF
S, 17 mg/L, PHO1
S, 10% total protein, PHO1
S, 160 mg/L, -MF
S, 180 mg/L, -MF
S, 20 mg/L, -MF
S, 30 mg/L, native
I, 0.25 mg/L
S, 670 mg/L, -MF
I, 15% total protein
S
259
260
261
262
263
264
265
266
267
268
269,270
S, 50 mg/L, -MF
S, 3 g/L, native
S, 240 mg/L, -MF
S, 4 mg/L, -MF
S, 5 mg/L, pre Mucor pusillus
rennin signal
S
S, 10 mg/L, PHO1
S, 30 mg/L, -MF
S, 170 mg/L, -MF
248
24,271274
275277
278
I, 10 g/L
S, 3 mg/L, -MF
I, 15 mg/L
S, 600 IU/mL, pre Mucor
pussils rennin
31,289
290
291
292
Vascular endothelial growth factor (VEGF165)
S, 40 mg/L, PHO1
293
S, 10 mg/L, -MF
279
280
281
282,283
18,48
284287
288
Viruses
A/VICTORIA/3/75 influenza virus
neuraminidase head domain
Bovine herpes virus-1 glycoprotein D
Dengue virus type 1 structural
gene recombinant E protein
Hepatitis B virus surface antigen
Hepatitis B virus surface
antigen-HIV gp41 epitope chimera
Hepatitis E virus ORF3
Human immunodeficiency
virus type 1 (HIV-1) envelope glycoprotein,
Polyomavirus large T antigen
Reovirus 1 core protein
Reovirus 1 protein
Vaccinia virus complement control protein
S, 3 mg/ml, -MF
294, 295
S, 20 mg/L, -MF
S, PHO1, prM virus signal sequence
296, 297
298
I, 400 mg/L
25, 299
I
I
gp120 (ENV) S, 20 mg/L, -MF
300
301
302
I, 0.5 mg/L
I, 0.8 mg/L
I
S, 3 mg/L, a-MF
303
304
305
41
I = Intracellular (with subcellular location), S = Secreted, S* = Secreted to plasma membrane. Amounts are highest reported for
particular protein. Signal sequences: -MF (S. cerevisiae -mating factor); PHO1 (P. pastoris acid phosphatase); SUC2
(S. cerevisiae invertase).
Volume 16, 2000

expression strains, and, as a result, a variety of
fed-batch and continuous culture schemes are
available (10,11). All schemes involve the initial
growth of strains in a defined medium on glycerol. During this period, growth is rapid but heterologous gene expression is fully repressed.
Upon depletion of glycerol, a transition phase is
initiated in which additional glycerol is fed to cultures at a growth-limiting rate. Finally, methanol,
or a mixture of glycerol and methanol, is fed to
cultures to induce expression. The time of harvest,
typically the peak concentration of a foreign protein, is determined empirically for each protein.
High-density fermentation of P. pastoris expression strains is especially attractive for the production of secreted proteins, because their
concentration in the culture medium should increase with cell density. Unfortunately, the concentrations of other cellular materials, particularly
proteases, increase as well. Three strategies have
proven effective in minimizing the proteolytic instability of foreign proteins secreted into the P.
pastoris culture medium. One is the addition of
amino acid-rich supplements, such as peptone or
casamino acids, to the culture medium which appear to reduce product degradation by acting as
excess substrates for one or more problem proteases (28). A second is changing the culture medium pH (28). P. pastoris is capable of growing
across a relatively broad pH range from 3.0 to 7.0
which allows considerable leeway in adjusting the
pH to one that is not optimal for a problem protease. A third is the use of a protease-deficient P.
pastoris host strain (10,11).
10. Expression of Protein

for Structural Studies
The P. pastoris yeast expression system has
gained widespread use for production of proteins
for structural studies. Proteins that are normally
secreted or require mammalian post-translational
modifications can not be produced in an active
form in E. coli. For these proteins in particular,
large quantities of active protein can be produced
in P. pastoris. Since P. pastoris grows rapidly on
inexpensive media similar to E. coli, it is feasible
to quickly and cheaply synthesize and examine
37
large numbers of mutant proteins suitable for
structure/function studies. The P. pastoris yeast
expression system offers key advantages relative
to other mammalian expression systems and to
Bacculovirus. In particular, P. pastoris grows in
minimal medium that can be modified for production of 15N-labeled proteins (37-41). Even triply
labeled (15N, 13C, 2H) proteins have been produced in this way (42,43). In some cases, the
yields of labeled proteins compete favorably with
those obtained from E. coli (4446). Another advantage of the P. pastoris yeast expression system is that glycosylated proteins can be prepared
in high yields and the sugars that are attached by
P. pastoris are of a simple high mannose form
(47,48). These sugars are easily removed by
cleavage with endoglycosidase H, leaving the
core GlcNac if advantage for X-ray crystallographic analysis where a homogeneous protein
sample is required (4950). Indeed, crystallography of glycosylated proteins can be extremely difficult if the protein is produced in mammalian
cells or in Bacculovirus because of incomplete removal of the complex sugars that these organisms
attach. A comprehensive list of heterologous proteins expressed in P. pastoris can be found in
Table 3 and at www.kgi.edu/html/noncore/program 4.htm#jc.
11. Pichia methanolica:

New Kid on the Block
Recently P. methanolica, another methylotrophic species, has been developed as a heterologous expression system (51,52). The essential features of an expression system have been developed
for this yeast, including, strains, expression vectors, transformation system (mostly via non-homologous recombination), a methanol-regulated
promoter for recombinant protein expression, and
fermentation techniques. A possible advantage
over P. pastoris is in fermentation; biomass generation during fermentation can use glucose instead
of glycerol and less methanol is required for the
induction of recombinant protein expression. The
main disadvantage of P. methanolica is that its
relatively new arrival as an expression tool means
that it is less well characterized, including optiVolume 16, 2000
38
mization of parameters for transformation, protein expression, and fermentation. Similar to the
situation with P. pastoris in which its use is covered by patents that are owned by RCT, patents
covering the use of P. methanolica are owned by
ZymoGenetics, Inc. of Seattle, WA. Although
use for academic research purposes is permitted,
a license is required for commercial use after an
initial one-year commercial evaluation period.
Also like P. pastoris, P. methanolica has been licensed to Invitrogen Corp., Carlsbad, CA for distribution to academic and commercial users. P.
methanolica strains, plasmids, and licensing information can be obtained from Invitrogen.
12. Summary
Based on available data, there is an approx 50
75% probability of expressing any protein of interest in P. pastoris at a reasonable level. The
biggest hurdle seems to be generating initial successthat is, expressing a specific protein at any
level. After success at this stage, there are welldefined parameters that can be manipulated to
optimize expression, and it is often at this stage
that attractive levels of expression are achieved.
Although there are relatively few examples of
expression of 10 g/L, there are many examples
of expression in the 1 g/L range, ranking the P.
pastoris expression system as one of the most productive eukaryotic expression systems available.
There are also examples of proteins that have been
successfully expressed in P. pastoris that were
completely unsuccessful in Baculovirus or S.
cerevisiae expression systems, making the P.
pastoris system an important alternative to have
available in the protein expression "toolbox." See
Table 3 for a comprehensive list of heterologous
proteins expressed in P. pastoris.
Acknowledgments
The preparation of this manuscript was supported
by Grant DK-43698 from the National Institutes of
Health and Grant ER20334 from the Department of
Energy, Office of Basic Energy Sciences (to
J.M.C.). We would like to thank Terrie Hadfield,
Oregon Graduate Institute, for expert assistance in
preparation of the manuscript, and Elizabeth
Cregg et al.
Komives, University of California, San Diego, for
information on heavy atom labeled protein.
References
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Foreign gene expression in yeast: a review. Yeast 8,
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3. Romanos, M. (1995) Advances in the use of Pichia
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L. A. (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic
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Recombinant Protein Expression

Recombinant Protein Expression in Pichia pastoris

Volume 16, 2000

Fig. 1. Results of Medline search conducted on

As a yeast, P. pastoris is a single-celled

In this review the basic aspects of the P.

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Recombinant Protein Expression

4. Secretion of Heterologous Proteins

*Northern Regional Research Laboratories, Peoria, IL.

in several instances (10,11). SMD1163 (his4 pep4

Recombinant Protein Expression

HIS4 and kanr

HIS4 and kanr

NotI sites for AOX1 gene

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Recombinant Protein Expression

9. Expression in Fermentor Cultures

Recombinant Protein Expression

S, 2.5 g/L, SUC2

I, 2.0 103 U/mg

S, 2.3 g/L, PHO1

S, 0.15 mg/L, PHO1

S, 1 mg/L, synthetic native, PHO1

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S, 120 mg/L, -MF

S, native and -MF

S, 11.5 g/L, native

S, 720 mg/L, PHO1

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Recombinant Protein Expression

S, 0.2 mg/L, native

S, 300 mg/L, -MF

I, S*, 1.5 g/L, SUC2

S, 1.5 g/L, -MF

S, 6.3 mg/L, -MF

S, 11.6 mg/L, -MF, PHO1

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S, 100 mg/L, -MF

S, 24 mg/L, 0.1 mg/L, -MF

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Recombinant Protein Expression

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Placental protein-14 (PP-14)

Vascular endothelial growth factor (VEGF165)

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Recombinant Protein Expression

10. Expression of Protein

11. Pichia methanolica:

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Recombinant Protein Expression

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47. McAlister, M. S., Davis, B., Pfuhl, M., and Driscoll,

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Recombinant Protein Expression

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Recombinant Protein Expression

novel, papain-related proteinases. Eur. J. Biochem.

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Pichia pastoris of the recombinant vaccine Gavac

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Recombinant Protein Expression