/REVIEW • © 1993 Nature Publishing Group http://www.nature.

com/naturebiotechnology

Recent Advances in the Expression of Foreign Genes

in Pichia pastoris
James M. Cregg*, Thomas S. Vedvick1 and William C. Raschke1
Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-1999. 1The Salk
Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA 92037-4641. *Corresponding author.

The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of
a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and
application of the system have improved its utility even further. These advances include: (1) methods for
the construction of P. pastoris strains with multiple copies of AOXI-promoter-driven expression cassettes;
(2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to
reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purifica-
tion of secreted products; and (5) detailed infonnation on the structures of N-linked oligosaccharides on
P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris
system are described and discussed.
s host organisms for the production of eukaryotic the understanding and application of the P. pastoris system. The
heterologous proteins, yeasts combine the molecular discussion focuses on vector and host strain construction tech-
genetic manipulation and growth characteristics of niques, strategies for the production of foreign proteins in fer-
prokaryotic organisms together with the subcellular mentor cultures, and the purification and characterization of
machinery for performing post-translational protein secreted products. Other aspects of the system have been cov-
modification of eukaryotes. The first yeast selected ered in previous reviews7- 13 •
for this purpose was Saccharomyces cerevisiae due
to the accumulated knowledge of its genetics and physiology and Construction of Expression Vectors
its acceptance as safe for human use through experience with the and Strains
organism in brewing and baking1 • However, S. cerevisiae has The primary features that make the P. pastoris expression
not proven to always be ideal as a foreign gene expression host. system unique are a direct consequence of the inherent tran-
The methylotrophic yeast Pichia pastoris is the most highly scriptional properties of the promoter most commonly used to
developed of a small group of alternative yeast species chosen control foreign gene expression. This promoter is derived from
for their perceived advantages over S. cerevisiae as expression the AOXJ gene of P. pastoris. The AOXJ gene was isolated by
hosts2 •3 • Two attributes were critical in its selection: the exist- searching a P. pastoris eDNA library for clones that were
ence of well-established fermentation methods and the presence expressed in methanol- but not ethanol-grown cells and then
of methanol-regulated promoters. During the 1970's, P. pastoris screening for methanol-regulated clones that encoded an amino
was investigated for potential use as a source of single-cell acid sequence identical to the known amino-terminal sequence
protein (SCP)4 • As a result, fermentation techniques were devel- of AOX 14 • Further studies of AOX genomic clones revealed that
oped for maintaining the organism in large-volume continuous the P. pastoris genome also harbors a second functional alcohol
culture and at cell densities in excess of 100 grams/liter dry cell oxidase gene, AOX21' . AOX2 encodes a protein that is 97%
weight. The growth medium, a defined mixture of salts, trace identical to and has approximately the same specific activity as
elements, biotin and carbon source, is inexpensive and can be that of AOXJ1'· 16 • However, in P. pastoris cells growing on
formulated free of toxins and pyrogens. Separately, biochemical
studies established that methanol metabolism required induction TABLE 1. Heterologous proteins produced In Plchla pastorls.
of a unique set of pathway enzymes'. From an expression system Percent of Amount
perspective, the most interesting of these was alcohol oxidase Protein Mode+ Protein (g/1) Reference
(AOX), the first enzyme in the methanol-utilization pathway. ~-galactosidase 20 ND 8
AOX is undetectable in cells cultured on carbon sources such as Thmor necrosis factor 25 8.0 28
Hepatitis B surface antigen 3 0.30 26
glucose, glycerol or ethanol, but constitutes up to 30% of total Superoxide dismutase 2 0.75 G. Holtz*
soluble protein in methanol-grown cells6. It was anticipated that Human interleukin-2 30 4.0 G. Davis*
AOX synthesis would be regulated at the transcriptional level Tetanus toxin fragment C 27 12 29
Pertactin (P69) 10 3.0 31
and that the promoter from this gene would be most useful for
controlling the expression of foreign genes. Under control of an Invertase s 80 2.5 23
Bovine lysozyme s 60 0.30 27
AOX promoter, foreign genes could be maintained in an "expres- Human lysozyme s ND 0.70 G. Davis*
sion-off'' mode on a non-methanolic carbon source to minimize Human serum albumin s ND 4.0 K. Sreekrishna*
selection for nonexpressing mutant strains during cell growth, Human epidermal
then efficiently switched on by shifting to methanol. With devel-
growth factor s 80 0.50 35
Mouse epidermal
opment and application of the P. pastoris system, these antici- growth factor s 80 0.45 25
pated advantages have been realized, along with others that were Human insulin-like
unanticipated. The P. pastoris expression system has now been
growth factor-1 s ND 0.50 R. Brierley*
Human interleukin-2 s ND 1.0 G. Veliceebi*
successfully utilized to produce a number of heterologous pro- Aprotinin analog s ND 0.80 24
teins at commercially interesting concentrations (see Thble 1). Kunitz protease inhibitor s ND 1.0 42
The purpose of this review is to highlight recent advances in +I=Intracellular; S=Secreted; *Personal communication.

BIOfTECHNOLOGY VOL. 11 AUGUST 1993 905

 Kb
• © 1993 Nature Publishing Group http://www.nature.com/naturebiotechnology
2.3-
1.3-
1.1-
2 3 4
FIGURE 1. Northern filter hybridization analysis of AOX steady
state mANA levels. Each lane contains 10 ll9 of total RNA
extracted from glucose (G)- or methanol (M)-grown cells of P.
pastorls. The labeled probes were P. pastorls genomic DNA
fragments encoding either AOX or glyceraldehyde-3-phos-
phate dehydrogenase (GAP). The AOX1 and GAP mRNAs are
2.3 and 1.1 kb, respectively.
methanol, AOXJ is responsible for all but a minor fraction of
total AOX message and protein. Thus, it is the promoter from
AOXJ that is incorporated into most P. pastoris expression
vectors.
After isolation of AOXJ, the presumption was confirmed that
its expression is regulated at the transcriptional level. Northern
blots such as that shown in Figure 1 demonstrated the tight
control that the cell exercises over this gene8 • In methanol-grown
cells, AOXJ message represents approximately 5% of total
polyA+ RNA but is undetectable in cells utilizing most other
carbon sources8 • Further investigations outlined the probable
regulatory systems that control AOXJ transcription' 7 • Like the
well-studied S. cerevisiae galactose- and ethanol-utilization-
pathway genes, AOXJ appears to be under the control of both a
general carbon catabolite repression/derepression mechanism
and a carbon-source specific induction mechanism. However,
AOXJ promoter regulation is unusual in that derepressing condi-
tions are necessary but not sufficient for significant transcription
of AOXJ. In carbon-starved cells, the most derepressing condi-
tions known for P. pastoris shake flask cultures, AOXJ message
and protein levels reach only 2% of those observed in methanol-
grown cells 17 •
The virtual requirement for methanol for transcription from
the AOXJ promoter is unique and has important implications for
production of heterologous proteins in large-volume high-den-
sity fermentor cultures. In such cultures, other expression sys-
tems require high concentrations of a repressing carbon source
during biomass generation to keep expression switched off. The
repressing substrate must then be removed prior to induction.
With P. pastoris cultures, high levels of repressing carbon
source are not needed to prevent transcription from the AOXJ
promoter. Therefore, during biomass generation, the concentra-
tion of repressing carbon source, typically glycerol, needs only
to be sufficient to meet the growth requirements of the cells.
Fermentor cultures can then be induced by simply allowing cells
to utilize the repressing growth substrate to exhaustion and then
adding methanol-procedures that are readily performed in
906 BIOITECHNOLOGY VOL. 11 AUGUST 1993
large-volume high-density cultures with existing fermentation
technology.
Basic techniques required for construction of P. pastoris
expression strains are generally similar to those used for S.
cerevisiae. These include: (1) classical-genetic manipulation
methods such as mutant isolation, complementation analysis,
backcrossing and spore analysis' 5 •18 ·' 9 ; and (2) molecular-genetic
methods of DNA-mediated transformation, gene targeting, gene
replacement and cloning by functional complementation15 ·2()-22 •
However, differences between this methylotrophic yeast and S.
cerevisiae have required development of procedures specific to
growth and manipulation of P. pastoris. The structure of a
typical P. pastoris expression vector is shown in Figure 2. The
vector, pA0815, is composed of AOXJ promoter and transcrip-
tional terminator fragments (5 'AOXJ and 3'AOXJ, respectively),
separated by a unique EcoRI site into which the foreign gene of
interest is inserted. The vector also contains the P. pastoris
histidinol dehydrogenase (HJS4) gene for selection in P. pastoris
his4 hosts, and sequences from E. coli plasmid pBR322.
The goal of most expression efforts in P. pastoris is the
secretion of the foreign protein. Secretion requires the presence
of a signal sequence to target the protein into the secretory
pathway. Constructions utilizing the native signal sequence of
the protein of interest are convenient in that they can be prepared
by inserting the entire coding sequence of the gene into the
EcoRI site of the expression vector. However, in many cases, P.
pastoris has not been able to efficiently utilize the native signal
sequence to direct secretion, e.g., invertase23 • As one alternative
signal, the 89 amino acid S. cerevisiae prepro alpha mating
factor (a-MF prepro) leader sequence has been effective at
directing secretion of several proteins from P. pastoris'-"·25 •
To maximize the stability of foreign protein production
strains, expression vectors are integrated into the P. pastoris
genome. As in S. cerevisiae, cleavage of a P. pastoris vector
within a sequence shared by the host genome stimulates homol-
ogous recombination events that efficiently target integration of
the vector to that genomic locus20•21 • For expression vectors such
as pA0815 shown in Figure 2, two integration options are avail-
able. The plasmid vector can be linearized by cutting at one of
several unique restriction sites in either HIS4 or AOXJ 5'
sequences to stimulate single-crossover type integration events.
Alternatively, the vector can be cut with Bglll to release its
expression cassette on a DNA fragment with AOXJ terminal
sequences which stimulate one-step gene replacement events at
AOXJ. The resulting strains are deleted at AOXJ which forces
them to rely on the transcriptionally weak AOX2 gene15 • These
strains metabolize methanol at a greatly reduced rate but some-
times express higher levels of foreign protein than wild-type
hosts, especially in shake flask cultures, e.g., !3-galactosidase17 ,
invertas&3 , and hepatitis B surface antigen26 •
Recently, methods to construct P. pastoris strains with multi-
ple copies of a heterologous gene expression cassette have been
described. Early P. pastoris expression strains were made with
only a single expression cassette copy' 7 •23 •26 •27 • Although several
of these strains produced commercially useful concentrations of
foreign protein, strains harboring multiple copies often (but not
always, see ref. 11) synthesize significantly higher levels of
protein. 1\vo approaches toward obtaining multi-copy expres-
sion strains have evolved. One approach is to identify multi-copy
strains that exist naturally at a frequency of a few percent within
transformed cell populations. Large numbers of individual
transformants are either directly screened for product levels by
SDS-polyacrylamide gel electrophoresis28 , immunoblotting29 •30 ,
or indirectly screened by colony "dot" blot hybridization for
transformants with multiple copies of the foreign gene31 • Using
this approach strains harboring up to 30 copies of an expression
 •
cassette have been isolated".
© 1993 Nature Publishing Group http://www.nature.com/naturebiotechnology

The second approach is to introduce multiple expression

cassette copies into a single vector prior to transformation''· 24 , as
diagrammed in Figure 2. The foreign gene is inserted into the
EcoRI site of pA0815 to create a vector such as pA0816. The
resulting single cassette vector is digested with Bglll and BamHI
to excise the expression cassette. The cassette is then reinserted
at the BamHI site of pA0816 to create a tandem repeat of the
cassette as in vector pA0817. This reinsertion process is repeated
to generate a series of vectors that contain increasing numbers of
cassettes. An advantage of this latter approach is that the task of
recovering each expression cassette from the final production
strain for sequence verification analysis is manageable. Multi-
copy expression strains of both types have proven to be stable
under the selective pressure of production in fermentor cultures.

Growth of R pastorls Expression Strains

Independently isolated P. pastoris strains transformed with
the same expression vector routinely display a range of product
levels. This clonal variation is observed even within collections
of transformants harboring the same number of expression cas-
settes. Thus, to find the best producer, it is necessary· to screen a
significant number of transformants. This has presented a prob-
lem in the past because levels of foreign protein obtained from
shake flask cultures of P. pastoris often did not accurately reflect
the levels observed from fermentor cultures and tended to be
disappointingly low. Potential reasons for this include: a lack of
pH control in typical shake flask cultures, inadequate aeration of
cultures, or inability to control feeding of carbon sources at
optimal (growth-limiting) rates. Th avoid the laborious alterna-
tive of testing each potential expression strain in fermentor
culture, simple small-scale shake tube and shake flask culture
conditions have been developed that result in product levels that
more closely resemble those expected in the fermentotJO. The
culture conditions include: medium buffering that is adequate
for the culture density and adjusted to a pH that minimizes FIGURE 2. Scheme for construction of vectors with multiple
proteolytic degradation of product, maximum aeration, and copies of a foreign gene expression cassette (from ref. 11).
addition of a modest amount of peptone or casamino acids which
may protect the product from proteolysis by a carrier mecha-
nism or may provide amino acids and energy for foreign protein
synthesis and secretion.
A hallmark of the P. pastoris system is the ease by which
expression strains scale up from shake-flask to large-volume
high-density fermentor cultures without loss of specific produc-
tivity. Considerable effort has gone into the optimization of
foreign protein production in :fermentors and as a result, a
variety of fed-batch and continuous culture schemes are now
availabl&2- 35 • Each takes advantage to varying degrees of the
ability to separately control cell growth and production in P.
pastoris. The most extreme example of this separation is
achieved in expression hosts in which the AOXJ gene has been
deleted. As diagrammed in Figure 3, cell mass is first generated
by culturing cells in a defined medium on glycerol. During this
period, growth is rapid but heterologous gene expression is fully
repressed. Upon depletion of glycerol, methanol is added to the
culture to induce production of the foreign protein with little
additional cell growth.
Although this two-step process has been effective in stimulat-
ing high levels of expression of some products, the time required
to achieve peak product concentrations is long, typically 150 to
200 hours after shift to methanol because of the low metabolic
activity of AOXJ-deleted hosts8•23 •26-28 • This long production
phase has been reduced to 50 hours or less without reducing
product concentrations through the use of wild-type hosts and
the application of one of several recently developed mixed-feed
fermentation strategies32- 35 • In essence, these strategies each FIGURE 3. Diagram depleting the general fermentation strat-
involve the addition of a transition phase between growth and egy for production of foreign proteins In P. pastorls.

BIO/TECHNOLOGY VOL. 11 AUGUST 1993 907

 • © 1993 Nature Publishing Group http://www.nature.com/naturebiotechnology
FIGURE 4. Polyacrylamide gel of S. cerevlslae Invertase
secreted from P. pastoris. The four right lanes each contain
5 1-11 of growth medium withdrawn at the indicated times
from a fermentor culture at a density of approximately
40 g (dry weight) per liter. Also shown are purified invertase
from P. pastorls (P.p.; 10 j.lg) and Invertase from S. cerevls/ae
(S.c.; 15 1-1g). The gel was stained with Coomassle Blue (from
ref. 23).
production phases in which cultures are primed for induction by
feeding glycerol at growth-limiting rates. The transition phase is
then followed by one of two modified production phase schemes.
In one, methanol is fed to the culture at a growth-limiting rate. In
the other scheme, the glycerol feed, started during the transition
phase, is continued along with methanol. With this mixed
glycerol-methanol feeding scheme, some expression strains
appear to be more metabolically active and to synthesize product
at a much greater rate than on methanol alone. The decision as to
which production scheme to employ, as well as specific parame-
ters within each scheme, are determined empirically.
High-density fermentationof 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.
In this environment, some secreted proteins are quite stable,
e.g., invertase2\ bovine lysozyme2\ where as others are signifi-
cantly degraded, e.g. EGP· 3S, HSA (K. Sreekrishna, personal
communication). Three strategies have proven effective in mini-
mizing the proteolytic instability of specific foreign proteins
secreted into the P. pastoris culture medium. The first is the
addition of amino acid-rich supplements such as peptone or
casamino acids to the culture medium which, as mentioned
above, reduce product degradation possibly by acting as excess
substrates for one or more problem proteases30 • The second is
changing the culture medium pH25 •35 • P. pastoris is capable of
growing across a relatively broad range of pH from 3.0 to 7.0
which allows considerable leeway in adjusting the pH to one
where degradation is significantly reduced. The third is the use
of a host strain in which the P. pastoris homologue of the S.
cerevisiae PEP4 gene has been deleted (M. Gleeson, personal
communication). PEP4 encodes a vacuolar protease that is
908 BIOffECHNOLOGY VOL. 11 AUGUST 1993
responsible for proteolytic activation of other vacuolar prote-
ases3•. Use of a mutant pep4 P. pastoris host improved the yield
ofiGF-1 approximately 50% in fermentorcultures (M. Gleeson,
personal communication). Further, experiments in which puri-
fied IGF-1 was incubated in broth from pep4 and wild-type
cultures demonstrated that the growth factor was more stable
in the pep4-strain broth which indicates that the improved
yield was a direct result of reduced proteolysis (M. Gleeson,
personal communication). Importantly, the three proteolytic sta-
bilizing strategies have displayed an additive effect when used in
combination.
Purification and Characterization of
Secreted Products
Yeast expression systems are particularly valued for their
ability to secrete heterologous protein products since passage of
proteins through the secretory pathway permits post-transla-
tional events such as proteolytic maturation, glycosylation and
disulfide bond formation to occur. A specific advantage of secre-
tion in P. pastoris is that since the organism secretes only very
low levels of native proteins, secretion of a foreign protein
becomes an effective purification step that separates product
from most other cellular components. This is dramatically dem-
onstrated in Figure 4, which shows a stained polyacrylamide gel
containing 5-~1 samples of fermentor broth from a culture of P.
pastoris secreting S. cerevisiae invertase23 • Virtually the only
material visible is invertase.
Despite the apparent purity of many foreign proteins secreted
from P. pastoris, further purification is almost always necessary.
The following scheme has proven to be an effective starting point
for the purification of a variety of secreted products. Cell-free
broth is diluted 1:1 (v/v) with 50 mM sodium acetate (NaAc)
pH 5. The diluted broth is then passed through a column filled
with a strong cation exchange resin. The column is washed with
the NaAc buffer and the secreted protein product is eluted with a
linear gradient ofO to 1M NaCl in NaAc buffer. At this stage of
purification, the protein is typically 90% pure or greater. A
sometimes-useful second step in purification is hydrophobic
interaction chromatography (HIC). Because the post-ion-
exchange column material is already in high salt, little treatment
is required to prepare it for application to a HIC column.
Additional salt ~S04) is added to the solution to achieve 40 to
50% saturation. The solution is then passed through a column
containing an appropriate HIC resin. Phenyl and butyl HIC
resins have been used successfully but this varies from protein to
protein. The product is eluted from the column by a decreasing
linear gradient from 40 to 0% ofN~S04 • After elution from the
HIC resin, the products generally have been greater than 95%
pure and after dialysis and lyophilization appear as a white fluffy
protein powder. Both ion-exchange and HIC chromatography
steps have proven effective and can be scaled up to accommodate
large volumes. Additional down-stream chromatography steps
may be needed to improve purity or to separate monomeric
forms from multimers of the same molecule.
The structure of carbohydrate added to secreted proteins is
known to be very organism specific37.38 • Furthermore, N-aspara-
gine-linked oligosaccharide structures on proteins secreted from
S. cerevisiae have been demonstrated to be exceedingly anti-
genic when introduced into mammals and as a result, the devel-
opment of foreign glycoprotein products from yeasts for use as
human therapeutics has been avoided. Comparison of S. cerevi-
siae invertase secreted from S. cerevisiae and P. pastoris has
revealed distinct differences between N-linked oligosaccharide
structures added to proteins secreted from these yeasts23 •39 • In
both species, the majority of N-linked oligosaccharide chains
are the high-mannose type38 •39 • However, the length of oligosac-
charide chains added to P. pastoris-secreted S. cerevisiae inver-
 • © 1993 Nature Publishing Group http://www.nature.com/naturebiotechnology
tase (P. pastoris invertase) is much shorter than those from S.
cerevisiae23•38 •39 • For P. pastoris invertase, all but a few percent of
chains are in the size range Mans- 14GlcN~ (Man = mannose;
GlcNAc = N-acetylglucosamine), whereas only about 20% of
S. cerevisiae chains are this size39-4 1 • Furthermore, analysis of P.
pastoris-secreted invertase and total P. pastoris glycoproteins
revealed that even the longest chains contain only approximately
30 mannose residues, and thus are significantly shorter than the
50 to 150 mannose residue chains typically observed on S.
cerevisiae glycoproteins38 •40 • As shown in Figure 4, the net result
is thatP. pastoris invertase is a relatively homogeneous family of
molecular mass 85 to 90 kD, whereas S. cerevisiae invertase is
much larger and more variable at 100 to 140 kD.
A second major difference became apparent upon determi-
nation of P. pastoris oligosaccharide structures. Oligosac-
charides from P. pastoris-secreted S. cerevisiae invertase were
purified and their structures assigned using one- and two-dimen-
sional 1H NMR spectroscopyn. Figure 5 shows the major P.
pastoris species identified along with those of representative S.
cerevisiae and mammalian high-mannose-type molecules. The
most striking difference is that glycans from P. pastoris-secreted
invertase do not have the terminal a 1,3-linked mannose residues
that are characteristic of S. cerevisiae core oligosaccharides.
This result is not specific to P. pastoris invertase, since a 1,3-
linked mannose is also missing from total P. pastoris oligosac-
charide preparations, and since activity for a1,3 mannosyl
transferase is undetectable in P. pastoris (R. Trimble, personal
communication). The significance of these observations is that
the terminal a 1,3-linkages on S. cerevisiae glycans are primar-
ily responsible for the highly antigenic nature of S. cerevisiae
glycoproteins and make S. cerevisiae secreted glycoproteins
unsuitable for use as therapeutic products (M. Romanos, per-
sonal communication). However, it should also be cautioned
that most of the oligosaccharide structures on P. pastoris-
secreted invertase are not the same as those on mammalian
glycoproteins and that neither the extent to which P. pastoris
structures are antigenic in mammals nor the rate at which pro-
teins displaying those structures are removed from circulation
has been determined40 •
Conclusion
P. pastoris has proven to be a powerful tool for production of
foreign proteins of commercial interest. Advantages of the sys-
tem include: (1) the AOXJ promoter which has transcription
characteristics useful for regulating heterologous protein
expression; (2) well-developed methods for classical- and
molecular-genetic manipulation of the organism; and (3) tech-
nology for the growth of expression strains in large high-density
fermentor cultures. These, along with improvements in its
manipulation for expression purposes, have moved P. pastoris
into the mainstream of foreign gene expression systems.
Acknowledgments
We thank the many employees at The Salk Institute Biotechnology/
Industrial Associates, Inc. (SffiiA), Phillips Petroleum Company and
Wellcome Research Laboratories who have contributed to the P. pastoris
expression system. We are especially indebted to those listed in the text
who shared unpublished results. We are grateful to Paula Schoeneck for
many helpful discussions and critical review of this manuscript, and to
James F. Cregg and Xuqiu Tan for computer graphics. We also wish to
acknowledge Phillips Petroleum Company and SffiiA for support in the
preparation of this review.
References
1. Hitzeman, R. A., Hagie, F. E., Levine, H. L., Goedde), D. V., Ammerer, G.
and Hall, B. D. 1981. Expression of human gene for interferon in yeast. Nature
293:717-222.
2. Buckholz, R. G. and Gleeson, M.A. G. 1991. Yeast systems for the commercial
production of heterologous proteins. Bio/Thchnology 9:1067-1072.
3. Romanos,M. A., Scorer, C. A. andClare,J. J. 1992. Foreign gene expression in
yeast: a review. Yeast 8:423-488.
4. Wegner, E. H. 1983. Biochemical conversions by yeast fermentation at high-cell
'
,?
o;
o;
1,6 Mllllllose
I, 3 MIIDilOse
T o; 1,2 MIIDilOse
N-Acetylglucoslllllillll
Mammalian
Hiah Mannoae
P.JMIIOril
FIGURE 5. Diagram of structure of selected N-llnked oligo-
saccharides on S. cerevlslae Invertase secreted by P. pas-
torls. Sugar and linkage type are Indicated In the upper left
comer. Open circles with dashed lines represent the most
frequent position observed for the last mannose on P. pas-
torls ollgoaaccharldes. Terminal a1,3 linked mannoses on
the S. cerevlslae oligosaccharide core structure are shown
enclosed In rectangles.
densities. U. S. Patent 44114329.
5. Veenhuis, M., van Dijken, J. P. and Harder, W. 1983. The significance of
peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv.
Microb. PbYsiol. 24:1-82.
6. Couderc, R. and Baratti, J. 1980. Oxidation of methanol by the yeast Pichia
pastoris: purification and properties of alcohol oxidase. Agric. Bioi. Chern.
44:2279-2289.
7. Cregg, J. M., Tschopp, J. F., Digan, M. E., Siegel, R., Craig, W., Velicelebi,
G, and Thill, 0. 1988. High-level expression and secretion of heterologous•
proteins from the methylotrophic yeast, Pichiapastoris, p. 48-49.1n: Advances
in Gene Technology: Protein Engineering and Production. Brew, K., Ahmad,
F., Bialy, H., Black, S., Penna, R. E., Puett, D., Scott, W. A., van Brunt, J.,
Voellmy, W., Whelan, W. J. and Woessner, J. F. (Eds.). IRLPress, Washington,
DC.
8. Cregg, J. M. and Madden, K. R. 1988. Development of the methylotrophic
yeast, PichiG pastoris, as a host system for the production of foreign proteins.
Dev. Ind. Microbiol. 29:33-41.
9. Digan, M. E., Tschopp, J., Grinna, L., Lair, S. V., Craig, W. S., Velicelebi, G.,
Siegel, R., Davis, G. R. and Thill, G. P. 1988. Secretion of heterologous
proteins from the methylotrophic yeast, Pichia pastoris. Dev. Ind. Microbiol.
29:59-65.
10. Cregg, J. M., Digan, M. E., Tschopp, J. F., Brierley, R. A., Craig, W. S.,
Velicelebi, G., Siegel, R. S. and Thill, G. P. 1989. Expression of foreign genes
in Pichi<l pastoris, p. 343-352. In: Genetics and Molecular Biology of Industrial
Microorganisms. Hershberger, C. L., Queener, S. W. and Hegeman, G. (Eds.).
American Society for Microbiology, Washington, DC.
11. Thill, G. P., Davis, G. R., Stillman, C., Holtz, G., Brierley, R., Engel, M.,
Buckholz, R., Kenney, J., Provow, S., Vedvick, T. and Siegel, R. S. 1990.
Positive and negative effects of multi-copy integrated expression vectors on
protein expression in Pichi<l pastoris, p. 477-490. In: Proceedings of the 6th
International Symposium on Genetics of Microorganisms, Vol. n. Heslot, H.,
Davies, J., Florent, J., Bobichon, L., Durand, G. and Penasse, L. (Eds.).
Societe Francaise de Microbiologie, Paris.
12. Vedvick, T. S. 1991. Gene expression in yeast: PichiG pastoris. Curr. Opinion
Biotechnol. 2:742-745.
13. Tschopp, J. F. and Cregg, J. M. 1991. Heterologous gene expression in mcthylo-
trophic yeast, p. 305-322. In: Biology of Methylotrophs. Goldberg, I. and
Rokcn, J. S. (Eds.). Butterworth-Heinemann, Stoneham, MA.
14. Ellis, S. B., Brust, P. F., Koutz, P. J., Waters, A. F., Harpold, M. M. and
Gingeras, T. R. 1985. Isolation of alcohol oxidase and two other methanol
regulatable genes from the yeast Pichia pastoris. Mol. Cell. Bioi. 5: llll-1121.
15. Cregg, J:M., Madden, K. R., Barringer, K. J., Thill, G. P. and Stillman, C. A.
1989. Functional characterization of the two alcohol oxidase genes from the
yeast Pichi<lpastoris. Mol. Cell. Bioi. 9:1316-1323.
16. Koutz, P., Davis, G. R., Stillman, C., Barringer, K., Cregg, J. and Thill, G.
810/TECHNOL.OGY VOL. 11 AUGUST 1993 909
 • © 1993 Nature Publishing Group http://www.nature.com/naturebiotechnology
Hannover, 19-21 October 1993
Central Forum
For
Biotechnology
BIOTECHNICA '93, which will take place from
19th - 21st October 1993 in Hannover/Germany,
offers valuable solutions for today and insights
into the future. It is the leading international
event for biotechnology.
The Exhibition
Approx. 450 exhibitors will showcase the latest
products and research trends in the following
fields of application: health care/pharmaceuticals,
environmental protection/recycling, the chemical
industry, the food industry, agriculture, bio
energy, raw materials production, and analysis.
The Congress
The Congress will open on 19th October 1993
with an international environmental symposium
dealing with the issues raised at the Earth Summit
in Rio de Janeiro.
The Congress will also focus on biotechnology
applications in environmental protection (19th
October), health/medicine (20th October) and food
production (21st October).
The Supporting Program
The Exhibition and Congress will be backed up by an
international program of seminars, symposia and
company presentations.
See you in Hannover! Further information is
available on request.
- ~!Q!~,~,tlo~~~~
TECHNICA B I 0 TEcH N 0 l 0 G y
HANNOVER 19- 21 OCTOBER 1993
For information, contact: Deutsche Messe AG, Messegelonde
D-3000Hannover 82, Telephone +49 511 89-3 8011/12, Telex 9 22728
Fax +49 5 11 89-3 80 03, Btx* 30143 #
(new postal code after 1st July: D-30521 Hannover)
trJ DEUTSCHE MESSE AG, HANNOVER/GERMANY
Write In No. 515 on Reader Service Card
910 BIOfTECHNOLOGY VOL. 11 AUGUST 1993
1989. Structural comparison of the Pichia pasroris alcohol oxidase genes. Yeast
5:167-177.
17. Tschopp, J. F. , Brust, P. F., Cregg, J. M., Stillman, C. A. and Gingeras, T. R.
1987. Expression of the lacZ gene from two methanol-regulated promoters in
Pichia pasroris. Nucleic Acids Res. 15:3859-3876.
18. Cregg, J. M. 1987. Genetics of methy1otrophic yeasts, p. 158-167. In: Proceed-
ings of the Fifth International Symposium on Microbial Growth on C I Com-
pounds. Duine, J. A. and van Verseveld, H. W. (Eds.). Martinus Nijhoff
Publishers, Dordrecht, The Netherlands.
19. Liu, H., Thn, X., Veenhuis, M ., McCollum, D. and Cregg, J. M. 1992. An
efficient screen for peroxisome-deficient mutants of Pichia pasroris. J. Bacte-
rial. 174:4943-4951.
20. Cregg, J. M., Barringer, K. J., Hessler, A. Y. and Madden, K. R. 1985. Pichia
pastoris as a host system for transformations. Mol. Cell. Bioi. 5:3376-3385.
21 . Cregg, J. M. and Madden, K. R. 1987. Development of yeast transformation
systems and construction of methanol-utilization-defective mutants of Pichia
pastoris by gene disruption, p. 1-18. In: Biological Research on Industrial
Yeasts, Vol. II. Stewart, G. G., Russell, I.. Klein, R. D. and Hiebsch, R. R.
(Eds.). CRC Press, Boca Raton, FL.
22 . Cregg, J. M. and Madden, K. R. 1989. Use of site-specific recombination to
regenerate selectable markers. Mol. Gen. Genet. 219:320-323 .
23. Tschopp, J. F.. Sverlow, G., Kosson, R ., Craig, W. and Grinna, L. 1987. High-
level secretion of glycosylated invertase in the methylotrophic yeast, Pichia
pastoris. Bio/Technology 5:1305-1308.
24. Vedvick, T., Buckholz, R. G., Engel, M., Urcan. M., Kinney, J., Provow, S.,
Siegel, R. S. and Thill, G. P. 1991. High-level secretion of biologically active
aprotinin from the yeast Pichia pastoris. J. Ind. Microbial. 7:197-201.
25. Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A.,
Payne, M. M., Sreekrishna, K. and Henwood, C. A. 1991. Production of mouse
epidermal growth factor in yeast: high-level secretion using Pichia pastoris
strains containing multiple gene copies. Gene 105:205-212.
26. 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., Thrregrossa, R., Velicelebi, G . and Thill, G. P. 1987. High-level
expression and efficient assembly of hepatitis B surface antigen in the methylo-
trophic yeast, Pichiapaswris. Bio/Technology 5:479-485.
27. 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., Velicelebi, G., Harpold, M.
M. and Thill, G. P. 1989. Continuous production of a novel lysozyme via
secretion from the yeast, Pichia pastoris. Bio/Technology 7:160-164.
28 . 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 of recombinant human tumor
necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Bio-
chemistry 28:4117-4125.
29. 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 Pichia pastoris
strains containing multiple tandem integrations of the gene. Bio/Technology
9:455-460.
30. Barr, K. A., Hopkins, S. A. and Sreekrishna, K. 1992. Protocol for efficient
secretion of HSA developed from Pichia pastoris. Pharm. Eng. 12:48-51.
31. 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 Bordetel/a pertussis pertactin (P69) from the yeast Pichia pas-
roris: high-level production and immunological properties. Vaccine 9:901-906.
32. Siegel, R. S. and Brierley, R. A . 1989. Methylotrophic yeast Pichia pastoris
produced in high-cell-density fermentations with high cell yields as vehicle for
recombinant protein production. Biotechnol. Bioeng. 34:403-404.
33. Brierley, R. A., Siegel, R. S., Bussineau, C. M., Craig, W. S., Holtz, G. C.,
Davis, G. R., Buckholz, R. G., Thill, G. P., Wondrack, L. M., Digan, M. E.,
Harpold, M. M.,l..air, S. V., Ellis, S. B. and Williams, M. E. 1989. Mixed feed
recombinant yeast fermentation. International Patent Application, Publication
No. WO 90/03431.
34. Brierley, R. A., Bussineau, C., Kosson. R .• Melton, A. and Siegel, R. S. 1990.
Development of recombinant Pichia pastoris expressing the heterologous gene
bovine lysozyme. Biochem. Eng. VI 589:350-362.
35. Siegel, R. S., Buckholz, R. G., Thill, G . P. and Wondrack, L. M. 1990.
Production of epidermal growth factor in melhylotrophic yeast cells . Interna-
tional Patent Application, Publication No. WO 90/10697.
36. Hirsch, H. H., Rendueles, P. S. and Wolf, D. H. 1989. Yeast (Saccharomyces
cerevisiae) proteinases: structure, characteristics and function, p. 134-200. In:
Molecular and Cell Biology of Yeasts. Walton, E. F. and Yarranton, G . T.
(Eds.). Black.ie and van Nostrand Reinhold, New York.
37. Goochee, C. F., Gramer, M. J., Andersen, D. C., Bahr, J. B. and Rasmussen, J.
R. 1991. The oligosaccharides of glycoproteins: bioprocess factors affecting
oligosaccharide structure and their effect on glycoprotein properties. Bio/Thch-
nology 9:1347-1355.
38. Kukuruzinska, M. A., Bergh, M . L. E. and Jackson, B. J. 1987. Protein
glycosylation in yeast. Ann. Rev. Biochem. 56:915-944.
39. Grinna, L. S. and Tschopp, J. F. 1989. Size distribution and general structural
features of N-linked oligosaccharides from the melhylotrophic yeast, Pichia
pastoris. Yeast 5:107-115.
40. Byrd, J. C., Threntino, A. L., Maley, F., Atkinson, P. H. and Trimble, R. B.
1982. Glycoprotein synthesis in yeast identificaiton of Man 8 GlcNAI::l as an
essential intermediate in oligosaccharide processing. J. Bioi. Chern. ZS7: 14657-
14666.
41. Trimble, R . B., Atkinson, P. H., Tschopp, J. F., Thwnsend, R. R. and Maley, F.
1991. Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted
by the rnethylotruphic yeast Pichia pastoris. J. Bioi. Chern. 266:22807-22817.
42. Wagner, S. L., Siegel, R. S., Vedvick, T. S., Raschke, W. C. and van Nostrand,
W. E . 1992. High-level expression, purification and characterization of the
Kunitz-type protease inhibitor domain of protease nexin-2/amyloid 13-protein
precursor. Biochem. Biophys. Res. Commun. 186:1138-1145.