GENE

ELSEVIER

Gene 190 ( 1997) 55-62

Strategies for optimal synthesis and secretion of heterologous proteins in

the methylotrophic yeast Pichia pastorid
Koti Sreekrishna *, Robert G. Brankamp, Keith E. Kropp, Dale T. Blankenship, Jiu-Tsair Tsay,
Phil L. Smith, Jonathan D. Wierschke, Arun Subramaniam, Lori A. Birkenberger
Hoechst

Marion

Roussel,

Inc., 2110 East Galbraith Road, Cincinnati,

OH 45215-6300,

USA

Received 8 March 1996; revised 14 June 1996; accepted 9 July 1996; Received by S.E. Hasnain

Abstract
Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia
using the methanol oxidase promoter. The factors that drastically influence protein production in this system include:
copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5- and
3-untranslated regions (UTR), translational start codon (AUG) context, A+ T composition of cDNA, transcriptional and
translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions,
and fermentation parameters. All these factors should be considered in designing an optimal production system. The inherent
ability of P. pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms
(which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the
production of MMP. However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

pastoris,

Keywords:
Alcohol oxidase promoter; Expression plasmid design; Transcriptional barrier; Synthetic gene; 5-Untranslated sequence;
mRNA secondary structure; Integrative transformation;
Gene dosage; Clonal variation; G418R selection; Growth and media
optimization; Secretion; Protease-deficient strain; Cell shrinkage; Apoptosis; Fermentation

1. Introduction
In recent years, several industrial yeasts, owing to
their robust growth and certain other unique characteristics, have been developed as recombinant host systems
for the commercial production of heterologous proteins
(Romanos et al., 1992). One such yeast, Pichiu pastoris,
* Corresponding
author. Tel. + 1 513 9484586; Fax + 1 513 9487345;
e-mail: kotisreekrishna@hmri.com
1Presented at the International Conference on Eukaryotic Expression
Vector Systems: Biology and Applications,
National
Institute
of
Immunology,
New Delhi, India, 4-8 February
1996.
Abbreviations:
aa, amino acid(s); AMF, d mating factor; AOXZ, gene
encoding
alcohol (methanol)
oxidase; B., BaciNus; BDNF, brainderived neurotrophic
factor; bp, base pair(s); Bs, Bacillus sphaericus;
BSP, Bs mosquitocidal
protein; cDNA, complementary
DNA; EGF,
epidermal growth factor; E., Escherichia; HSA, human serum albumin;
MMP, matrix metalloproteinase(s);
Mut, methanol utilization phenotype; nt, nucleotide(s);
P., Pichia; PAGE, polyacrylamide
gel electrophoresis; S., Saccharomyces; SDS, sodium dodecyl sulfate; TIMP- 1,
tissue inhibitor of MMP; R, resistance/resistant;
TNF, tumor necrosis
factor; Ubi, ubiquitin; UTR, untranslated
region(s); YP, yeast extract
and peptone.
0378-l 119/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved
PZZ SO378-1119(96)00672-S

has proven to be an excellent host for the production

of both secreted and intracellular proteins (Romanos
et al., 1992; Sreekrishna and Kropp, 1996).
P. pastoris is a methylotrophic yeast. It can grow on
methanol as a sole carbon and energy source. It possesses
a highly inducible methanol utilization pathway.
Methanol oxidase, the first enzyme of the pathway,
accounts for up to 35% of the total protein in cells
grown on limited amounts of methanol. The enzyme is
undetectable in cells grown on glucose, ethanol or
glycerol (Wegner and Harder, 1986). This highly inducible and stringently regulated methanol oxidase gene
(AOXl) promoter has been used to construct expression
vectors for the production of heterologous proteins in
P. pastoris. Using this system, a wide variety of proteins
have been produced with varying degrees of success
(Romanos et al., 1992; Sreekrishna, 1993; Sreekrishna
and Kropp, 1996). Although the final yield of a protein
is greatly influenced by its inherent properties, nevertheless, the yield can be significantly enhanced by manipulation of the factors that influence gene expression and
product stability (Sreekrishna, 1993). Strategies and

K. Sreekrishna et al. J Gene 190 (1997) 55-62

56

prospects for enhancing the production level of proteins

in the P. pastoris expression system are examined here.

2. Results and discussion

2.1. Autonomous replication versus chromosomal
integration of the expression cassette
The preferred mode of expression in P. pastoris is by
chromosomal integration using one of the integrative
plasmids (e.g., see Fig. 1). There are several advantages
for using integrative transformation
which include:
expression cassette stability, generation of multicopy
strains (see Section 2.4), control of site of integration
(HIS4 or AOXZ loci), and ability to engineer different
modes of integration (with or without the eviction of

the AOXI coding sequences, see Section 2.3) using

appropriately cleaved DNA. Plasmids based on autonomous vectors such as PHIL-Al (Fig. 2) are of low copy
number, unstable, and invariably integrate at one or
more of the chromosomal loci, namely AOXl, HIS4, or
ARSI. Thus, the use of autonomous plasmids necessitates a secondary screen to isolate stable integrants. The
single most significant advantage of autonomous plasmids in protein production is their ability to transform
P. pastoris spheroplasts at a high frequency as compared
to linear integrative plasmids (Sreekrishna et al., 1987).
2.2. Site of integration of the expression cassette
Both AOXl and HIS4 sites have been used successfully for expression. We have noticed occasional loss of
the lacZ expression cassette at the HIS4 locus due to
gene conversion between chromosomal mutant copy of
the his4 and the good HIS4 gene of the expression
cassette. Thus, it would appear that the AOXl locus is
the preferred site.
2.3. Methanol utilization phenotype of the host: Mut
or Mut Transformation of a P. pastoris his4 strain with a
linear DNA whose ends are homologous to the 5 and

PHIL-D 1
7749 bp
BglII(5319)

Fig. 1. Plasmid PHIL-Dl, an E. cobP. pastoris shuttle vector, with

sequences required for selection in each host. The left half of the plasmid is a portion of pBR322, from ClaI (at nt 7676 on the map) to
PvuII site [modified to BglII (at nt 5319 on the map)]. This segment
of pBR322 contains the ampicillin-resistance-encoding gene (ApR) and
the origin of replication (ColEl ori). The EcoRI site in this segment
has been eliminated. The DNA elements comprising the rest of the
plasmid are derived from the genome of P. pastoris, except for short
regions of pBR322 used to link the Pichia sequences. The P. pastoris
elements in the plasmid are as follows: (1) 5-AOXI, approx. 1000 bp
of the alcohol oxidase promoter. The AOXl sequences following the
nt A of the ATG start codon have been removed by BAL 31 treatment
and the synthetic linker 5-GGAATTC added to generate a unique
EcoRI cloning site. (2) AO-t, approx. 300 bp of the AOXl transcriptional terminator sequence. (3) P. pastoris histidinol dehydrogenaseencoding gene, HIM, contained on a 2800 bp fragment to complement
the defective his4 gene in the P. pastoris strains such as GS115,
SMD1168, etc. (4) Region of 3-AOXI DNA approx. 650 bp, which
together with the 5-AOXI region is necessary for the site-directed
displacement of AOX1-coding sequences (see Fig. 5). The unique Sac1
(at nt 209 on the map) and SalI (at nt 2875 on the map) sites present
in the plasmid can be used for site-directed integration of the entire
plasmid into the AOXI and HIS4 loci of P. pastoris, respectively.

PHIL-AI
6400 bp

//

Fig. 2. Plasmid PHIL-AI, an E. coli-P. pastoris shuttle vector, with

sequences required for selection and autonomous replication in each
host. The left half of the plasmid is a modified portion of pBR322
containing the Aps gene and the ColEl ori. The DNA elements comprising the rest of the plasmid are derived from the genome of P.
pastoris, except for short segments of pBR322 used to link the yeast
sequences. The various P. pastoris sequences are as follows: (1) 5AOXI, approx. 750-bp portion of the AOXI promoter with the EcoRI
(at nt 337 on the map) engineered as previously described (Fig. 1). (2)
AO-t and HIS4 are the same as in Fig. 1. (3) PARSI, approx. 190-bp
segment of a P. pastoris autonomous replication sequence. The unique
San (at nt 2885 on the map) site can be used to direct the integration
of PHIL-AI into the HIS4 loci of P. pastoris.

51

K Sreekrishna et al. / Gene 190 (1997) 55-62

Nael(6916)

NaeI(4330)

Fig. 3. Plasmid PHIL-D2. This plasmid has a 458-bp DNA containing

the fl-ori inserted beween the original DraI sites of pBR322. The Not1
sites at nt 8 and 5337 are used for site-directed displacement of AOXIcoding sequences. The other details are the same as described for
PHIL-Dl (Fig. 1).

_--

2.4. Gene dosage: exploiting the clonal variation of

expression

There are numerous examples in which a single copy

of the expression cassette is sufficient for optimal production (Cregg et al., 1985, 1987; Sreekrishna et al.,
1990, 1993) and deliberately increasing the copy number
has no significant effect on production. Yet in other
cases, multiple copies of the expression cassette (> 10)
are a must for high level expression. A dramatic effect
of copy number on protein production in P. pastoris is
seen in the production of human TNF, tetanus toxin
fragment C, Bordetella pertussus pertactin P69, and
mouse epidermal growth factor, EGF (Sreekrishna et al.,
1988a; Romanos et al., 1991; Clare et al., 1991a,b;

PHIL-D7
9442 bp
BglII(6573)

3 regions of the AOX chromosomal locus (e.g., BgflI

digested PHIL-Dl (Fig. 1), PHIL-D7 (Fig. 4) or pPIC9
(Scorer et al., 1994) or NotI-digested PHIL-D2 (Fig. 3))
results in the site-specific eviction of the AUXl structural
gene (Cregg et al., 1987; Sreekrishna et al., 1988b;
Sreekrishna, 1993) as illustrated in Fig. 5. The eviction
of the AOXI occurs at a frequency of l-5 per 20 His+
transformants.
A OX-deleted
clones show a slower
growth phenotype
(Mut -) on minimal methanol
medium, as compared to AOXl intact clones which
have phenotypically
normal growth characteristics
(Mut ) on minimal methanol medium.
For intracellular expression, it is preferable to use
Mut - cells because they will have a lower level of
alcohol oxidase protein and the expressed protein can
be more readily purified. For secretion, either one of
Mut+ or Mut- constructs can be used. We have not
seen any significant difference between these two types
of host cells in the secretion of HSA (Barr et al., 1992).

Ss110875)

A.

5 AOXl

3 AOXl

B.

Fig. 4. Plasmid PHIL-D7. This shuttle plasmid contains the kanamytin-resistance-encoding

gene ( KmR) and is useful for selection of
multicopy transformants based on increased level of resistance to
G418. Unlike the commonly used vectors such as PHIL-Dl and
PHIL-D2, we have designed PHIL-D7 such that it has a unique AsuII
site at nt position 934 (the second AsuIl site present in the 3-AOXI
has been eliminated). Therefore, the sequence 5-TTCGAAACG can
be added immediately upstream from the start codon (ATG) of the
gene of interest and an EcoRI site can be added downstream from the
stop codon. The modified gene can then be inserted between the AsuII
(at nt 934 on the map) and EcoRI (at nt 944 on the map) region 5TTCGAAACGAGGAATTC of PHIL-D7. Thus, the modified gene
will have an identical 5-VTR to that present in AOXl (see Section 2.5).
The other details are the same as described for PHIL-Dl (Fig. 1).

5 AOXl

5 AOXl

3 AOXI

3 AOXl

Fig. 5. Site-specific eviction of AUXI by gene replacement. (A) BgflIdigested DNA derived from PHIL-Dl expression plasmid used for
transformation. Gene X is the gene of interest cloned at the EcoRI
site of PHIL-Dl. (B) P. pastoris chromosomal AOXl locus. (C)
Chromosomal structure resulting from the replacement of the entire
AUXl locus by the transforming DNA (shown in A).

58

K. Sreekrishna et al. / Gene 190 (1997) 55-62

Sreekrishna, 1993). In some rare instances, an increase

in copy number has a negative effect on the production
level (Thill et al., 1990).
Thus, the effect of gene copy number on expression
is unpredictable. A practical solution to this problem is
to examine the production level as a function of gene
dosage. The spheroplast method of transformation of
P. pastoris results in transformants with a wide range
of copy numbers (Sreekrishna et al., 1988a; Clare et al.,
1991a,b; Sreekrishna, 1993), as was previously found in
the case of S. cerevisiae (Sreekrishna and Dickson,
1985). Analysis of as few as 100 individual clones for
protein production is generally adequate to arrive at a
good producer. If other methods of transformation (e.g.,
LiCl method or electroporation) which do not give rise
to multicopy transformants at a high frequency are
used, then more efficient screens, such as colony hybridization (or dot blot analysis) with DNA probes, or
selection based on increased level of resistance to the
antibiotic (e.g., G418) as a function of gene dosage can
be employed (Scorer et al., 1994).
Alternatively, multicopy constructs of the expression
cassette can be obtained by transformation with DNA
concatamers or using specially constructed multicopy
vectors, such as pA0856 and ~A081 5 (Thill et al., 1990;
Sreekrishna and Kropp, 1996). This is not a method of
choice as it permits production analysis over only a
narrow range of gene dosage.
2.5. mRNA 5- and 3-UTR
The nt sequence and the length of the 5-UTR can be
detrimental to optimal protein production. The leader
length of the highly expressed AOXZ mRNA is 114 nt
long and the sequence is A + U rich. For optimal synthesis of heterologous proteins, it is essential to maintain
the 5-UTR as closely as possible to that of the AOXlmRNA. Ideally, it is preferable to make it identical to
that of the AOXI-mRNA. The expression level of HSA
is increased over 50-fold by adjusting the 5-UTR to be
identical to that of the AOXl-mRNA (Sreekrishna et al.,
1990; Sreekrishna, 1993).
We have recently constructed a plasmid (PHIL-D7)
which can be used to readily make 5-exact constructs
(see Fig. 4).
2.6. Translation start codon AUG context
An AUG sequence should be avoided in the 5-UTR
to ensure efficient translation of mRNA from the actual
translation initiation site. The mRNA secondary structure around the AUG start codon may be adjusted, so
that AUG is relatively free of secondary structure as
predicted by the RNA-fold analysis (PC/gene Software,
Intelligenetics, Mountain View, CA, USA). This can be
accomplished by redesigning the initial portion of the

coding sequences with alternate codons (Sreekrishna,

1993).
2.7. A + T composition
Genes with high A+T nt content are not transcribed
efficiently due to premature termination (Romanos
et al., 1992). One such sequence that has been identified
to block transcription
in P. pastoris is 5ATTATTTTATAAA
found in HIV-gp120, and when
this stretch is altered to S-TTTCTTCTACAAG,
the
premature termination is abolished (Scorer et al., 1993).
Because there are many yet unidentified AT-rich
stretches that act as transcriptional terminators, a general strategy would be to redesign the genes so as to have
an A+T content in the range of 30-55%. Using this
approach, it has been possible to construct strains for
efficient production of tetanus toxin fragment C (Clare
et al., 1991a) and BaciZlus sphaericus mosquitocidal
toxins, BSPl and BSP2 (Sreekrishna, 1993; Sreekrishna
et al., 1993). For designing synthetic genes, use of
P. pastoris preferred
codons (Sreekrishna,
1993;
Sreekrishna and Kropp, 1996) is recommended.
2.8. Secretion signal
Secretion is the preferred mode of protein production
due to ease of product recovery. For a protein that is
not normally secreted, it may be difficult or even impossible to cause secretion. Likewise, for normally secreted
proteins such as HSA and growth hormones, it is
impossible to express them intracellularly in the soluble
form. Choice of secretion signal is rather arbitrary. In
several instances such as HSA (Barr et al., 1992),
invertase (Sreekrishna et al., 1987; Tschopp et al., 1987)
and bovine lysozyme (Digan et al., 1989) the native
signal peptide is adequate. We have recently found that
the native secretion
signals present
in matrix
metalloproteinases-1,2,
3,9 (MMP-1, 2, 3,9) and tissue
inhibitor of matrix metalloproteinases- 1 (TIMP- 1) are
functional in the P. pastoris system.
If a native secretion signal sequence is not available,
then a signal sequence based on that found present in
the S. cerevisiae invertase or the a mating factor (AMF)
can be used. The pre-pro AMF signal works very
efficiently especially for the secretion of smaller-sized
products such as aprotinin (Wagner et al., 1992), EGF
(Siegel et al., 1990; Clare et al., 1991b), thrombomodulin
fragment (White et al., 1994), blood factor XII (White
et al., 1994) a fragment of amyloid B-protein (Van
Nostrand et al., 1994), antibody single-chain Fv fragment (Ridder et al., 1995) and ghilanten (Brankamp
et al., 1995).
In making protein fusions to the AMF signal, it is
prudent to retain the Glu-Ala spacers adjacent to the
KEX2
protease
cleavage
site (..V-S-S-L-E-Lys-

K. Sreekrishnaet al. / Gene 190 (1997) 55-62

Arg-KEX2G1u-Ala-DApGlu-Ala-vDAP
fused protein). The
presence of the Glu-Ala spacers helps to alleviate the
steric interference by the fused protein resulting in an
efficient cleavage of the pro-sequence by the P. pastoris
KEXZlike protease (Thill et al., 1990). The Glu-Ala
spacer sequence is subsequently cleared by a diamino
peptidase (DAP) to yield the protein of interest free of
additional N-terminal aa residues. In spite of taking this
precaution, we have noticed that the pro-sequence is
not cleaved when the AMF signal is fused with human
BDNF (approx. 14 kDa). In this situation BDNF is
secreted into medium as a 26-30 kDa product. This is
the expected result if the pro-portion of AMF signal is
not cleaved and is heterogenously glycosylated at the
multiple glycosylation sites present in the AMF propeptide.
A secretion vector PHIL-Sl, based on the P. pastoris
PHOl (acid phosphatase) secretion signal (designed by
Rich Buckholz) is available, although its efficacy as such
in heterologous protein secretion is not documented.
However, recently Laroche et al. (1994) have demonstrated high level secretion of a tick anticoagulant peptide in P. pastoris, using a hybrid PHOl secretion signal
peptide containing a KEX2 cleavage site.
Over-expression of ubiquitin (Ubi) seems to enhance
the secretion of a human leukocyte protease inhibitor
in S. cerevisiae (Chen et al., 1994). We have recently
established that Ubi is a normal secreted component of
P. pastoris and we have proposed a role for Ubi in
protein secretion (Sreekrishna et al., 1995). We predict
that the over-expression of Ubi in P. pastoris may also
enhance heterologous protein secretion. A P. pastoris
host strain that over-expresses Ubi is not currently
available. However, it can be engineered using one of
the available Ubi cDNAs. Also, a Ubi expression plasmid can be introduced into any P. pastoris strain using
one of the dominant selection markers for transformation, such as invertase, SUC2 (Sreekrishna et al., 1987),
G418R (Sreekrishna et al., 1984) or zeocinR (available
from Invitrogen, San Diego, CA). Thus, the effect of
Ubi over-expression on product secretion can be tested
in any of the existing production strains. A note of
concern may be warranted while using zeocinR for
selecting P. pastoris transformants. Since zeocin is a
strong mutagen, cells challenged with zeocin on a complex medium may acquire some undesirable mutations
that may interfere with growth and thus reduce the
overall productivity under the growth conditions in a
fermentor.
2.9. Product stabilization
Proteolytic stability of secreted proteins can be
improved by altering the pH of the media. The recommended pH range for experimentation is between 2.8
and 6.5. The product stability is further enhanced by

59

addition of Casamino acids or YP to the media (Siegel

et al., 1990; Clare et al., 1991b; Brierley et al., 1994;
Barr et al., 1992; Sreekrishna, 1993). For example,
secretion of HSA was significantly improved by raising
the pH of the medium from 5.2 to 6.0 and the yield was
further enhanced by addition of YP (Sreekrishna et al.,
1990; Sreekrishna, 1993). Production of mouse EGF
was favored at pH 3.0 in the presence of Casamino acids
(Clare et al., 1991b). Addition of Casamino acids to
stabilize the product is preferable to YP, because the
peptide components of YP (such as bovine collagen
fragments) can interfere in product analysis and recovery. Addition of 5 mM EDTA to the medium also
improves product stability. It should be noted that
medium manipulation
can significantly alter the
profile of protein components, such that previously
unnoticed proteins can become evident. For example,
we have noticed that addition of EDTA causes the
accumulation of a protein of approx. 50 kDa in the
Pichia broth. This protein has the aa sequence
DIIWDYSSEKIMGVNLGGWL...,
which matches
closely the exe+ 1,3-glucanase
of S. cerevisiae
(Muthukumar
et al., 1993) and Candida albicans
(Chambers et al., 1993).
Product stability can also be enhanced using proteasedeficient strains such as SMD1168 (his4, pep4),
SMDl165 (his4, prbl) and SMDl163 (his4, pep4, prbl).
These strains have disruption in the genes encoding
protease A (PEPI) and/or protease B (PRBZ) (M.A.
Gleeson, personal communication).
These proteasedeficient strains have been invaluable in the production
of insulin-like growth factor- 1 (Brierley et al., 1994) and
ghilanten (Brankamp et al., 1995).
2.10. Expression of matrix metalloproteinases

We recently encountered an unexpected problem in

the production of human matrix metalloproteinases,
MMP-1, 2, 3, and 9. These enzymes in their natural
host are synthesized as pre-pro-proteins, secreted as proenzymes and eventually activated to mature enzymes
(Matrisian, 1990). In contrast to this, the MMPs produced in all three P. pastoris strains (SMD1168, GSl15
and KM71), were highly degraded. All the strategies
discussed above (Section 2.8) for stabilization failed to
stabilize these products. Furthermore, MMP-3 expression also caused significant modification of cells as
evidenced by accumulation of shrunken cells with lower
Over-expression
of
MMP-3
buoyant
density.
(Stromelysin-1 ) is known to cause apoptosis in the
mammary epithelial cells (Boudreau et al., 1995). Thus,
it is presumable that production of MMP-3 may be
causing apoptosis of P. pastoris cells. The effects noticed
with MMP-3 expression were also associated with
MMP-1 expression, although to a lesser extent. Based
on their ability to bind gelatin-Sepharose, we were able

K. Sreekrishna et al. 1 Gene 190 (1997) A-62

60

to purify sufticient quantities of MMP-2 and MMP-9 in

a single step for further analysis. The N-terminal aa
sequence of these products revealed that the pro-peptide
was absent in the majority of the purified preparation,
thus suggesting excessive proteolytic processing.
The instability problem of MMPs was partially overcome by coexpression of tissue inhibitor of matrix
metalloproteinases, TIMP-1. A very significant improvement in the accumulation of pro-protein was noticed in
the case of MMP-9. This observation is consistent with
the fact that TIMP-1 is known to bind tightly with the
pro-form of MMP-9. In contrast to this, TIMP-1
interacts only with the mature forms of MMP-1, 2 and
3 (Wilhelm et al., 1989). However, this is not a desired
approach for production of MMPs, because the MMPs
complexed with TIMP-1 cannot be readily dissociated.

12

345678

910

-250-

-6O-42-

-22-17-

2. Il.

Consequences of over-production of TIMP-1

Over-production of TIMP-1 in P. pastoris results in

both qualitative and quantitative changes in the secreted
proteins (Fig. 6). The effect was most significant with
respect to a particular protein(s) that migrated as a
diffuse doublet band in the 18-22 kDa region on
SDS-PAGE. This product which accumulated at nearly
a gram per liter level was purified by RF-HPLC
(reverse-phase high-performance
liquid chromatography) and sequenced. The N-terminal aa sequence of the
purified material ADYMCHMAC?GLAIYGAWEC
GPEAGPFDSECLLATD
(C refers to a possible Cys)
does not show any significant homology to any of the
previously characterized proteins in the aa sequence
data base.
The stabilization effect of TIMP-1 on P. pastoris
secreted proteins may be useful for enhancing the accumulation of heterologous proteins produced in this
system.
2.12. A new strategy for fermentation
Considerable time is expounded on setting up a
fermentor run. It is highly desirable to reap as many
cycles of product production as possible for each
start-up. Continuous fermentation on methanol, which
works well for biomass production (Wegner, 1983, 1990)
is largely not applicable for recombinant protein production, especially with Mut - strains, because they
grow poorly on methanol. Use of methanol+glycerol
mixed feed restores the cell productivity; however, the
optimal level of protein expression is not achievable due
to partial repression of the AOXI promoter by glycerol
(Sreekrishna et al., 1988b, 1989).
In search of carbon sources that support growth but
do not repress the methanol induction of AOXI promoter, John A. Cruze and W.D. Prevatt at Phillips
Petroleum Company found sorbitol and alanine as two

-6Fig. 6. Electrophoretic analysis of secreted proteins in the culture

medium from select clones of P. pastoris strain SMD1168 transformed
with the TIMP-1 expression plasmid. Transformants were hrst screened
for resistance to > 2 mg G418/ml, as previously described (Scorer et al.,
1994). Ten G418R clones were tested for protein secretion in BMMY
medium (buffered minimal methanol medium supplemented with YP;
Sreekrishna and Kropp, 1996) as per the protocol described previously
(Barr et al., 1992). Approx. 100 nl equivalent of the extracellular
medium from the induced cultures was subjected to PAGE on 4-20%
gradient gel (purchased from Novex, San Diego, CA, USA) under
reduced (P-mercaptoethanol) and denaturing (SDS) conditions in
Trisglycine buffer, pH 8.3. Proteins were visualized by staining with
Coomassie brilliant blue G-250. The migration position of the standards (in kDa) is indicated on the left margin of the gel. The arrow
points to the diffuse doublet protein band that is present at high levels
in a majority of the clones examined.

good substrates (personal communication). We have

recently employed sorbitol + methanol mixed feed batch
fermentation to accomplish several cycles of MMP-2
production in a Biostat-B (Braun) bench top fermentor.
In this approach, after recovery of 90% of the fermentor
sample from the first fermentation cycle, the fermentor is
rebooted with an appropriate volume of fermentor
growth medium containing sorbitol as carbon source
and supplemented with 0.1% yeast extract and 0.2%
peptone. After growth for 16-20 h, a fed batch fermentation with sorbitol + methanol nutrient feed is continued
for another 72 h for MMP-2 production. The whole
process of product recovery and rebooting the fermentor
can be repeated as many times as one desires. By this
approach, using a 4 1 fermentor with one start-up, we
were able to generate 25 1 of MMP-2 broth in less than
4 weeks. This approach is also useful for Mut cells,
because it reduces overall methanol consumption, as
most of the growth is supported by sorbitol; and methanol can be added to the sorbitol feed at any desired
point to initiate induction of expression.

K. Sreekrishna et al. / Gene 190 f 1997) 55-62

3. Conclusions and future perspectives

Many of the problems encountered in protein expression can be overcome by due consideration of the factors
that influence protein expression. Almost every expression project has given some new insight into the intricacies of the system. In spite of due consideration and
careful planning of an expression strategy, there are
certain proteins which simply cannot be expressed in
this system. Now that the expression system is readily
available from Invitrogen (San Diego, CA), hundreds
of investigators around the world are currently exploring
the system and their results will undoubtedly provide
new insights as well as expand our knowledge base
about the utility and limitations of the system.
Several improvements have recently been made to the
P. pastoris expression vectors by Invitrogen (San Diego,
CA). These include: (i) vectors for producing proteins
with epitope tag (useful for in situ protein localization),
(ii) smaller size vectors with zeocinR selection marker
(for ease of manipulation as well as a new dominant
selection marker for transformation),
and (iii) vectors
to provide a histidine tag (His,, to aid in ready purification of the expressed protein). We have previously
noticed that the addition of 8 aa C-terminal tags to
TNF to aid in its purification, rendered the tagged
proteins completely insoluble under non-denaturing conditions, whereas the untagged TNF was completely
soluble (Sreekrishna et al., 1988b). Thus, as tag enthusiasts, we should beware that a tag in some instances can
alter the property of a protein and bring about an
unintended and an undesirable end result.

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