Advances in the use of Pichia pastoris

for high-level gene expression

Mike Romanos
Glaxo Wellcome, Beckenham, UK
The Pichia pastoris system has the potential for very high level production
of foreign proteins. This, together with the recent availability of this system
in kit form, has changed Pichia from being an expression system with only
specialized biotechnological applications to one that is widely used for rapid
expression in the laboratory. The development of G418 selection protocols has
simplified the rapid isolation of multicopy transformants efficiently expressing
intracellular proteins. In addition, several strategies are now also available
for optimizing secretion, such as the generation of clones with progressively
increasing vector copy number, expression screening in microtitre plates, and
minimizing proteolysis by a number of techniques.
Current Opinion in Biotechnology 1995, 6:527-533
Introduction
In the past few years, the Pichia pastoris expression system
has rapidly achieved much wider use thanks to increasing
awareness of the early successes and to its sale in kit form
by Invitrogen (San Diego, USA). From being a specialist
system, used primarily by yeast groups in biotechnology
companies, it is now a mainstream expression host
used alongside Escherichia coli, Saccharomyces cerevisiae, and
baculovirus. This was clear from the diverse interests of
delegates at a recent Pichia meeting (San Diego, USA,
1994) organized by Invitrogen.
Pichia is an industrial methylotrophic yeast initially
chosen for production of single-cell protein because of its
ability to grow to very high cell density in simple defined
media. From this basis, a highly efficient expression
system was designed using the methanol-inducible
alcohol oxidase 1 (AOX1) promoter and vectors that
integrate into the Pichia genome [1]. Several examples
show that Pichia can routinely achieve percentage yields
(5-40% of cell protein) much higher than baker's yeast,
and often equivalent to E. coli or baculovirus [1,2].
Additionally, scale-up of Pichia culture to high cell
density is simple and has resulted in enormous yields on a
volumetric basis (e.g. >12 gl-I for tetanus toxin fragment
C [3] and >3 gl-1 secreted human serum albumin [HSA]

[4]).
Although most Pichia expression vectors are very similar,
different strategies can be employed because a selection
of host phenotypes and chromosomal sites of integration
is available. This gives greater scope for optimizing
expression, but has caused some confusion, particularly

among users unfamiliar with yeast genetics. In this

review, I attempt to clarify the choices that are available
and give suggestions as to when they should be used. I
also discuss some of the most recent developments and
results with the Pichia system, and provide an update on
some of the many Pichia-derived products in commercial
development or about to reach the market.

Pichia: advantages and disadvantages

Pichia has the following main advantages: first, extremely
high yields of intracellular proteins; second, very high
levels of secretion into an almost protein-free medium;
third, ease of fermentation to high cell density; and
fourth, genetic stability and scale-up without loss of
yield. It is proving valuable in producing large amounts
of protein for analytical studies; one interesting recent
application is in efficient in vivo isotopic labelling of
proteins for N M R [5]. The system is almost certainly
the simplest of any to scale up, a feature that makes it very
attractive for rapid production of biological products for
clinical trials.
Like any other expression system, however, Pichia is
no panacea, and examples of low yields or failure
of expression are also accumulating, though many
remain unpublished. Probably the commonest problem
encountered is proteolysis of secreted polypeptides
[1], though a number of ways of overcoming this
have become available. Another common problem is
inefficient secretion of complex foreign proteins (e.g.

Abbreviations
AOXl--alcohol oxidase 1; EGF-~epidermal growth factor; HSA--human serum albumin; IGF-l--insulin-like growth factor 1;
MutS--methanol utilization slow; PHO-1--acid phosphatase 1.

Current Biology Ltd ISSN 0958-1669

527

528

Expressionsystems
Notl
Avrl I

(a)

Nod
Avrl I
EcoRI i
SnaBI i

(b)

EcoRli
SnaBl!
Ncol I
BamHl[

Ncol I
BamHI [

Sad

Sac[

AOXI

Ap

Be/It,~

AOXIt

AOXI

pPIC3
7768 bp

pPIC3K
9009 bp

Ap

HIS4

AOXIt

HIS4

kan~'

Bgl~l
BglU
3'

AOXI

3' AOXI

(r) 1995CurremOpinionil~Biotechnology

HIV-1 gpl20 [6]). Despite many examples of yields

in the grammes per litre range, heterologous secretion
is more demanding than intracellular expression and not
guaranteed to work. Finally, some genes do not give any
detectable protein, often because yeast transcriptional
terminators result in truncated mRNA. The problem
was first described for the highly AT-rich tetanus toxin
gene in S. cerevisiae and solved by gene synthesis to
increase the GC content [2]. The same problem has
been seen in Pichia and solved in the same way (e.g.
with the Bacillus sphaericus Bsp2 insecticidal toxin [P1]
and with HIV-1 Env [6]). It is noteworthy that the Env
DNA, which is not AT-rich, is e~ciently transcribed in
S. cerevisiae.

Pichia vectors and strains

As Pichia has no stable episomal vectors, integrating
vectors are employed. All use HIS4 as a selectable marker
and have the same general organization, as exemplified
by pPIC3 (Fig. 1). Some of the most useful vectors
and their properties are listed in Table 1. The most
commonly used host strain is GSl15 (his4), but the
more recently available protease-deficient strains (e.g.
SMDl168; his4, pep4) are finding increasing use in
reducing proteolytic cleavage of secreted proteins. KM71
(his4, aoxl) and other strains that have an inactive A O X I
gene, are fundamentally different in that they grow very
slowly on methanol as a carbon source, for example,
during induction (i.e. they have a Muts phenotype,
rather than the Mut + phenotype of GSl15).
Expression vectors are directed to integrate into the
Pichia genome in one of two ways, depending on
where the DNA is cut before transformation (see
Table 2). Digestion to give a DNA fragment with
homology to AOX1 at both ends leads to replacement

Fig. 1. Two typical Pichia expression

vectors, pPIC3 and pPIC3K. (a) Vector
pPIC3 comprises a S' AOX1 region including promoter, cloning site polylinker,
AOXI terminator (AOX1 t), HIS4 marker,
3' AOX1 region and ampicillin-resistance marker (Ap). (b) Vector pPIC3K,
in addition to the above, contains the
kanamycin-resistance (kan r) marker for
G418 selection of multicopy transformants.

of genomic AOX1 by the fragment (i.e. transplacement)

and generates a Muts recombinant strain. Linearization
of the vector, by cutting either 5' to the AOX1 promoter
or within the HIS4 marker, directs integration of the
plasmid at the homologous sites in the genome. The Mut
phenotype of the latter type of transformant is dictated
by the host strain used.
Some unexpected features of Pichia transformations
increase the number of types of transformant and thus
the potential for confusion. With integration at AOX1
or HIS4, multicopy transformants (up to 10) can arise
from repeated recombination events. On the other
hand, transplacement would be expected to yield only
single-copy Mut s transformants; however, a detailed
analysis has revealed three sources of heterogeneity
among the transformants [3]. First, a high proportion
of 'transformants' contain no vector, express no foreign
protein and probably represent his4 gene conversions.
Second, only 5-30% are true transplacements (Muts),
the remainder have integrations at AOX1 or HIS4
and are Mut +. Finally, 1-10% of Mut s transformants
have up to 30 integrated copies of the transplacing
fragment as tandem head to tail repeats that probably
arise by a mechanism involving in vivo ligation [3].
The Mut + population also contains multicopy clones.
Although transplacement usually requires the laborious
sphaeroplast transformation technique because of low
frequencies, it yields a highly divergent population of
transformants, which is useful for detailed optimization
studies. Also, it seems to be the only way of obtaining
very high copy number transformants.

Gene dosage is critical for maximal expression

In some of the earliest studies, expression of 13galactosidase and hepatitis B surface antigen was not

High-level expression in Pichia pastori$ Romanos

Table 1. Pichia expression vectors.
Vector
name

Unique cloning
sites

Selection
markers

Comments

References

Intracellular production
pPIC3

BamHI, NcoI*
SnaBI, EcoRI,
Avrll and Nott

HIS4

Polylinker vector

[I 2 *]

:~PIC3K

BamHI, Ncol*
5naBI, EcoRI
Avrll and Nott

HIS4 and kan r

Polylinker vector with G418selection for multicopy clones

[12 ]

pHIL-DI

EcoRI

HIS4

One of the first basic vectors

[9]

pHIL-D2

EcoRI

HIS4

Contains fl ori and Notl

sites for transplacement

[a]

pHIL-D3

Asull and EcoRI

HIS4

Contains fl ori and native Asull site for

unaltered AOX1 5' untranslated region

[a]

pHIL-D4

EcoRI

HI54 and karl r

G418-selection of
multicopy clones

[a]

pHIL-D5

EcoRI

HIS4 and kar~

Contains fl ori, Nofl sites for

transplacement and G418-selection
for multicopy clones

[a]

pHIL-D7

Asull and EcoRI

and

Contains fi ori, a native Asull

cloning site, Nolt sites for
transplacement, and uses G418selection for multicopyclones

[a]

)AO815

EcoRI

Contains fl ori and a BamHI site for

generation of multicopy expression units

[1,16]

HIS4

kanr

HIS4

in vitro

Secretion
~HIL-SI

Xhol, EcoRI, 5mal

and BamHI

HIS4

Contains Pichia PH01 signal and fl ori

[a]

~)PIC9

Xhot, 5naBI,
EcoRI, Avrll and Noll

HIS4

Contains a 5. cerevisiae
a-factor leader

[12"]

pPIC9K

Xhol*, 5naBI,
EcoRI, Avdl and Notl

HIS4 and kanr

Contains a S. cerevisiae et-factor leader,

with G418-selection for multicopy clones

[12"']

pYAM7SP6

5tul, EcoRI, Bglll

Notl, Xhol, 5pel and
BamHI

HIS4

Contains a hybrid Pichia Phol signal with a

Kex2 endopeptidase cleavage site

[5"]

*The cloning site is not unique, thus three-way ligations are used. [a] K Sreekrishna, K Kropps, personal communication. (For further
details contact K Sreekrishna, Marion Merrell Dow Inc, 2110 East Galbraith Road, PO Box 156300, Cincinnati, Ohio 45215, USA.)

improved by increasing vector copy number [7,8]. In

numerous subsequent examples, however, the isolation
of multicopy integrants has resulted in dramatically
higher yields [3,9-11]. In a detailed study of tetanus
toxin fragment C, our group [3] showed that expression
was correlated with copy number (1-14 copies), whereas
site of integration and Mut phenotype had, at most,

only a minor effect on yield. This is perhaps surprising

because the Mut phenotype affects both the growth rate
during induction and the accumulation of large amounts
of endogenous alcohol oxidase. Mut s strains may yield
a higher proportion of correctly folded product in
situations where folding is rate-limiting (e.g. in the case
of hepatitis B surface antigen [8]).

529

530

Expressionsystems
Table 2. Types of Pichia transformant.
Host
strain

Vector digest
to direct
integration*

Resulting His+
transformants

GSl15
(Mut+)

Sad
(AOX1
integration)

Vector is integrated 5' to the genomic

AOXI gene, leavingthe AOXI gene
undisrupted (i.e. Mut*phenotype)

High-frequency transformation using

either sphaeroplasting or electroporation.
Ideal for routine use. M 00% of
transformants express protein. Multi-copy
integrants (up to 10 copies)
arise at low frequencies

Vector is integrated within genomic

his4 locus. AOXI gene
undisrupted (i.e. Mut+
phenotype)

High-frequencytransformationusing
either sphaeroplastingor electroporation.
-100% of transformantsexpress protein.
Note potential to generate His+ 'pop-outs'
lacking foreign geneL Multi-copyintegrants
(up to 10 copies) arise at low frequency

Bglll fragment replaces genomic

AOX1 gene, generating Mut s
phenotype. (Only 5-25% of
transformants are of this type, the
remainder are mainly AOXt or
HIS4 integrants li.e. Mut +
phenotype])

Low-frequency transformation, preferably

using the sphaeroplast method. This is best
method for multi-copy clones, yielding a l - I 0%
frequency and up to 30 copies. Generates a
heterogeneous pool of Mut s and Mut +
transformants, including some
non-expressers

Same transformants, as
GSI 15, except all are Mut s
because host AOXI gene
is already disrupted.

Higher transformation frequency than

GSI I 5, especially with electroporation

Sa~ and Stul

(HIS4 integration)

Bgnlt
(AOX1

transplacement)

KM71
(Muts)

5acl or 5all/Stul

Comments

*These sites are common to all vectors and can be used generally, unless present in the foreign gene. *The vector pHIL-D2 has Not1
sites in place of Bglll and can be used when the foreign gene contains Bglll sites. ~;Thisproblem does not often occur in small-scale
cultures.

The extremely high level of alcohol oxidase (5-30%)

expressed from A O X I in induced wild-type Pichia
had suggested that one copy of an A O X I expression
vector would produce maximal rnRNA levels. In a
recent paper, m R N A levels were analyzed from a series
of transformants containing increasing copy numbers
(1-12) of an HIV-1 Env expression vector [12]. The
Env m R N A level increased progressively with copy
number up to the maximum number tested; at a single
copy of the vector, it was two- to threefold less than
A O X 1 mR.NA, but with greater than three copies it
exceeded A O X 1 mRNA. A progressive increase was also
seen with fragment C, suggesting that transcription is, in
general, limiting in Pichia containing only a single copy
of the vector and that it is sensible to routinely maximize
copy number. Despite this, other factors, such as protein
stability, must have a major effect because final yields
of different proteins vary greatly, even with multicopy
clones. For example, 12 copies of Env vector yielded
2.5% of total cell protein, whereas 14 copies of fragrnent
C vector yielded 27%.
Aside from gene dosage, a critical factor that has
long been recognized to affect induction efficiency
is aeration of Pichia cultures. This results from the

tendency of cultures, especially Mut + strains, to become

oxygen-limited in shake-flask inductions, and it probably
explains the consistently large increase (e.g. 5-10-fold) in
yield that is observed when switching to fermenters.

Secretion in Pichia
Many users have been attracted to Pichia by the very
high reported levels of protein secretion in high-density
cultures, such that the product can comprise >80% of
the protein in the medium. Yet, secretion is complex
and is dependent not only on factors such as gene
dosage and Mut phenotype, but also on other factors
that affect the yield and quality of product (e.g. signal
sequence, processing, proteolysis and glycosylation).
Perhaps the most contentious issue, as demonstrated by
the lively debate at the recent meeting on Pichia in San
Diego, is Mut phenotype. Because secretory vesicles
in S. cerevisiae localize to the bud, it had been widely
believed that secretion could only occur in dividing
cells. Secretion of mouse a-amylase from S. cerevisiae has,
however, been shown to be as efficient in non-dividing

High-level expression in Pichia pastoris Romanos 531

as dividing cells [13]. The group at SIBIA Inc (La
Jolla, California) had favoured Mut + strains and have
used these in many examples of high-level secretion
(e.g. epidermal growth factor [EGF], insulin-like growth
factor-1 [IGF-1], aprotinin, etc. [14,P2,P3"]). Even so,
results with HSA [4] and murine EGF [15] demonstrate
that MutS strains can also yield high levels. Many more
recent successful examples utilize either Mut + or Mut s
strains.
In the case ofgene dosage, agreement is absolute that an
optimal, rather'than maximal, copy number is usually
required. In many cases, secretion efficiency has been
improved with several vector copies [P2,P3]. Examples
also exist where the maximum copy number tested was
optimal (e.g. with murine EGF [15]). With bovine
lysozyme, however, increasing the copy number from
one to three reduced the level of secreted product
[16], and for HIV gp120, a copy number of greater
than one reduced secretion and increased accumulation
of intracellular products [6]. It would appear that less
efficiently secreted proteins are likely to block the
secretory pathway at higher expression levels.
Several foreign proteins have been efficiently secreted
using their native signal peptide (e.g. HSA, where a
strain containing three copies of the gene, expressed from
a modified A O X 2 promoter, gave a staggering yield of
10 g1-1 [4,P4]). With bacterial a-amylase, however, the
yield using a yeast signal was two- to threefold higher
[17], and in general, yeast signals are more likely to
be successful [2]. Secretion of EGF and routine EGF
using the S. cerevisiae a-factor leader was shown to be
highly efficient, and analysis of the product showed
authentic processing at the Kex2 endopeptidase cleavage
site [15,P2]. The vectors pPIC9 and pPIC9K, which
contain the ct-factor leader sequence with convenient
cloning sites [12], have recently become available
from Invitrogen and are now widely used. Recent
successful examples using the or-factor leader include the
following: single-chain Fv antibody fragments [18]; a
9 kDa thrombomodulin fragment [19]; blood factor XII
[19]; a fragment ofamyloid ~-protein [20]; oncostatin M
(SJ McAndrew et al., abstract, Current Topics in Gene
Expression Systems: Pichia pastoris, San Diego, USA,
October 1994) coffee-bean ct-galactosidase (A Zhu, LF
Kimball, Current Topics in Gene Expression Systems:
Pichia pastoris, San Diego, USA, October 1994), and
cathepsin B (JS Mort et al., abstract, Current Topics in
Gene Expression Systems: Pichia pastoris, San Diego,
October 1994). An alternative signal sequence, that of
the Ih'chia acid phosphatase 1 (PH01) gene, is used in the
vector pHIL-S1, which is also available from Invitrogen.
Studies using S. cerevisiae have shown that the particular
yeast signal peptide used can affect efficiency of secretion
[2]. Recently, a synthetic hybrid signal based on Phol,
with an additional 19 residues, including a Kex2 cleavage
site, has been found to improve secretion of tick
anticoagulant protein and some other proteins two- to
threefold in Pichia [5]. Even so, it is not clear that

general rules concerning the signal peptide can be

applied to any protein.
The problem of proteolytic instability in the medium
has been encountered with several proteins secreted from
Pichia. It has been seen in shake flasks, but usually appears
to be far worse in fermenters, because of the higher
concentration ofproteases or the different medium used.
Three different approaches have been used successfully to
overcome proteolysis [1]: adding amino acid or peptide
supplements to the growth medium, buffering the pH
of the medium to a value where degradation is reduced
(e.g. pH 3), or using protease-deficient host strains. In
the case of IGF-1, no protein was produced unless the
medium was buffered to pH 3, but a 50% increase was
then achieved using a pep4 strain grown in a medium
buffered to pH 3 [P3].
S. cerevisiae has been avoided as a host for the development of human therapeutic glycoproteins because
yeast-derived glycoproteins are antigenic and frequently
hyperglycosylated (i.e. they contain extensive outer
chain mannose units [50-150 residues] that can mask
function). Pichia-derived invertase is, however, not
hyperglycosylated and has an outer-chain length of
8--14 units, compared with >50 in S. cerevisiae [21].
Bulk Pichia glycoprotein was found to be less frequently
hyperglycosylated, to have a shorter outer-chain length
(<30 residues), and to lack the highly antigenic terminal
al,3-mannose linkages present in S. cerevisiae [22,23].
Nevertheless, it should be emphasized that glycosylation
in Pichia and that in mammals is not identical, and it
is not known how antigenic Pichia glycoproteins are
or how the pharmacological properties of heterologous
products might be affected.

For the development of vaccines, totally authentic

glycosylation may be unnecessary, provided the proteins
elicit a protective immune response. Even so, results with
two viral glycoproteins, HIV1 gp120 and Epstein-Barr
virus gp350, were not promising. HIV-1 gp120 was
hyperglycosylated [6], whereas gp350 was less highly
glycosylated than the S. cerevisiae-derived material (CA
Scorer, personal communication), but neither of these
proteins was recognized by antibodies raised against
the native protein. In contrast, the Bm86 cattle tick
membrane glycoprotein, secreted using the invertase
signal peptide, was not hyperglycosylated and formed
immunogenic particles which partially protected cattle
against challenge [24].

Strategies for expression

For intracellular expression, it would seem reasonable
to isolate transformants with maximum vector copy
number. A G418-selection protocol using vectors containing the Tn903 kanr gene (e.g. pPIC3K) has recently
been described [12]. Using this procedure, multicopy
clones (5-10 copies) can be isolated, even following

532 Expressionsystems
electroporation, which had been thought to yield
only single-copy transformants. This greatly simplifies
the rapid isolation of multicopy clones and may be
considered the method of choice for general laboratory
use. In practice, this method works best using the KM71
strain and Sad-digested vector because of the higher
transformation frequencies that are attainable. Where
very high copy numbers (10-30) are required, however,
it then appears that transplacement using the sphaeroplast
transformation method must be used. Transformants can
subsequently be screened for high copy number 'jackpot
clones', either Muts or Mut +, using a rapid DNA
dot-blot method [11]. Precise copy numbers should be
determined using quantitative DNA dot blots [3].
For secretion, initial studies could be carried out with
single-copy transformants, but it would be preferable
to start with a series with different copy numbers.
Such a series could be created in several ways. A range
of mukicopy transformants, isolated by G418-selection
[12 ] or DNA dot blot [11], could be analyzed for
copy number [3]. An alternative method developed by
SIBIA is to utilize a plasmid that can be used to generate
multi-cassette vectors with up to eight copies in vitro
[1,16]. This method has the disadvantage that several
DNA cloning steps are required, but the advantage that
the copy number is determined before transformation.
Any of these approaches can be carried out in either
Mut + or Muts strains for comparison.
An alternative empirical approach to optimization
has been used for tick anticoagulant protein [5"].
Transplacement was used to generate a heterogeneous
pool of transformants and the level of secreted product
from 91 Muts and Mut + clones was tested in inductions
in microtitre plates. Eleven transformants were further
evaluated in shake-flask inductions and one chosen for
fermenter optimization, giving a a final product level of
1.7 gl-I of product. This method could be used in other
cases where a simple method exists for quantitating the
product.
Pichia transformants are generally tested in fermenters
as soon as possible because shake-flask inductions are
sub-optimal. Mut + and Mut s strains each have their
advantages: Mut + strains are less likely to become
poisoned by methanol, whereas Mut s strains are less
likely to become oxygen-limited. Although a matter
of debate, reports exist of induction using Mut s that
is equally rapid (48h) as that using Mut + strains
[3,10,11,15]. Because the increase in yield in going
from shake-flask to fermenter inductions is not always
predictable [11], it may be prudent to select several
transformants before proceeding to detailed fermenter
optimization.

Conclusions
The number of different proteins being expressed in
Pichia is expanding rapidly, and results from ongoing

studies will increase our knowledge of the capabilities

and limitations of this expression system. The more
wide adoption of Pichia, however, will require additional
refinements (e.g. more auxotrophic and mutant strains,
alternative selection markers, a drug-selection system
that shows greater dose-dependence than G418, vectors
for simukaneous expression of two proteins and alternative promoters to AOX1). These refinements, many of
which should be available shortly, will enable the I~'chia
system to gain some of the flexibility of S. cerevisiae.
Pichia is already widely accepted as an important
biotechnological host organism, and we are now at the
exciting stage of observing products moving through to
clinical trials and beyond. IGF-1 and HSA should soon
be marketed products for the treatment of amylotrophic
lateral sclerosis and as a serum replacement, respectively.
Numerous cytokines, vaccines and other biological
products are under development, and a Cuban group
has developed a hepatitis B vaccine that is currently
being sold in South America.

Acknowledgements
I would like to thank my colleagues in the field, especially Jeff
Clare, Koti Sreekrishna, Jim Cregg, Rich Buckholz, David Higgins, Mick Hunter and Bennet Cohen, for continually sharing
information with me.

References and recommended reading

Papers of particular interest, published within the annual period of
review, have been highlighted as:

of special interest
,
of outstanding interest
I.

Cregg JM, Vedvick TS, Raschke WC: Recent advances in the

expression of foreign genes in Pichia pastoris. Biotechnology
1993, 11:905-910.

2.

Romanos MA, Scorer CA, Clare IJ: Foreign gene expression in

yeast: a review. Yeast 1992, 8:423-488.

3.

Clare Jl, Rayment FB, Ballantine SP, Sreekrishna K, Romanos

MA: High-level expression of tetanus toxin fragment C in
Pichia pastoris strains containing multiple tandem integrations
of the gene. giotechnology 1991, 9:455-460.

4.

Barr KA, Hopkins SA, Sreekrishna K: Protocol for efficient

secretion of HSA developed from Pichia pastoris. Pharm Eng
1992, 12:48-51.

5.

Laroche Y, Storme V, De Meutter J, Messens l, Lauwereys M:

High-level secretion and very efficient isotopic labelling of tick

anticoagulant peptide (TAP) expressed in the methylotrophic
yeast. Pichia pastoris. Biotechnology 1994, 12:1119-1124.

A rapid microtitre plate expression screen is employed to obtain several

efficient TAP-secreting transformants from a pool for further optimization.
The authors achieved very high level secretion using a hybrid Phol signal
peptide containing a Kex2 cleavage site. Also reports an efficient method
for ~SN and 13C isotopic labelling of a protein that can then be used to
determine the solution structure by NMR.
6.

Scorer CA, Buckholz RG, Clare JJ, Romanos MA: The

intracellular production and secretion of HIV-1 envelope
protein in the methylotrophic yeast Pichia pastoris. Gene 1993,
136:111-119.

High-level expression in Pichia pastoris Romanos

7.

Cregg JM, Madden KN: Development of transformation systems

and construction of methanol-utilisation-defective mutants of
Pichia pastori$ by gene disruption. In Biological Research on
Industrial Yeast vol 2. Edited by Stewart GG, Russell I, Klein
RD, Hiebsch RR. Boca Raton: CRC Press; 1987:1-18.

display library and their production in the yeast Pichia

pastori$. Biotechnology 1995, 13:255-260.
The ct-factor leader vector, pPICg, is used to direct expression, allowing
secretion of functional antibody single-chain Fv fragment at >100mgl-1
in P. pastoris.

8.

Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig

WS, Buckholz RG, Madden KR, Kellaris PA, Davies GR et al.:
High-level expression and efficient assembly of hepatitis B
surface antigen in the methylotrophic yeast Pichia pastori$,
Biotechnology 1987, 5:479-485.

19.

9.

Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P,

Fish W, Motohiro F, Holden K, Phelps D, Wood P, Parker
K: High-level expression, purification, and characterisation
of recombinant human tumour necrosis factor synthesised
in the methylotrophic yeast Pichia pastorb. Biochemistry,
28:411 7-4125.

10.

SreekrishnaK, Potenz RB, Cruze JA, McCombie WR, Parker

KA, Nelles L, Mazzaferro PK, Holden KA, Harrison RG, Wood
PJ et al.: High level expression of heterologous proteins in
methylotrophlc yeast Pichia pastori~ ] Basic Microbiol 1988,
28:265-278.

11.

RomanosMA, Clare JJ, Beesley KM, Rayment FB, 8allantine

SP, Makoff AJ, Dougan G, Faitweather NF, Charles IG:
Recombinant Bordetella pertussi$ pertactin (P69) from the
yeast Pichia pastoris:, high-level production and immunological
properties. Vaccine 1991, 9:901-906.

ScorerCA, Clare JJ, McCombie WR, Romanos MA, Sreekrishna

K: Rapid selection using G418 of high cofly number
transformants of Pichia pastori$ for high-level foreign gene
expression. Biotechnology 1994, 12:181-I 84.
Polylinker vectors for intracellular expression (pPIC3 and pPIC3K) and
n-factor secretion vectors (pPIC9 and pPIC9K) are constructed with
and without the kanamycin-resistance marker for G418-selection. The
authors describe a rapid method for selecting multicopy transformants
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The 8m86 membrane glycoprotein from gut epithelial cells of the cattle
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in immunization experiments.

Patents

of special interest
of outstanding interest

P1.

Sreekrishna K, Prevatt WD, Thill GP, Davis GR, Koutz P,

Barr KA, Hopkins SA: Production of Bacillus entomotoxins in
methylotrophic yeast. 1993 EP0586892A1.

P2.

SiegelRS, Buckholz RG, Thill GP, Wondrack LM: Production

of epidermal growth factor in methylotrophic yeast cells. 1990
WO90/10697.

P3.

Brierley RA, Davis GR, Holtz GC: Production of insulinlike growth factor-1 in methylolrophic yeast cells. 1994
US5,324,639.
These authors use in vitro ligation to generate a multicopy o.-factor
expression vector for IGF-1 secretion. Secretion levels increase progressively with copy number, up to six copies. Because of proteo)ysis,
no product is detected, unless the induction medium is buffered to pH 3.
A further 50% increase is observed when using a pep4 protease-deficient
strain.
,,

Paifer E, Margolles E, Cremata J, Montesino R, Herrera L,

Delgado J-M: Efficient expression and secretion of recombinant
alpha amylase in Pichla pastoris using two different signal
sequences. Yeast 1994 10:1415-1459.
Improved secretion efficiency and product yield (from 0.9 g1-1 to 2.5 gl-])
is achieved by replacing the bacterial signal peptide sequence with that
from S. cerevisiae SUC2.

P4.

18.

M Romanos, Glaxo Wellcome, Langley Court, Beckenham, Kent

BR3 3BS, UK.

17.

Ridder R, Schmitz R, Legay F, Gram H: Generation of rabbit

monoclonalantibody fragments from a combinatorial phage

Miura M, Ishida, Y. Oi H, Murakami K, Nakagawa Y, Kawabe

H: Mutant AOX2 promoter, microorganism carrying same,
method of preparation thereof and production of heterologous
protein using such microorganism. 1992 EP92105201.5.

533