Indian Journal of Biotechnology

Vol 2, October 2003, pp 477-493

Evaluation of Yeast as an Expression System

M W Nasser, V Pooja, M Z Abdin and S K Jain*
Centre for Biotechnology, Jamia Hamdard, New Delhi 110 062, India
Received 20 March 2002; accepted 5 December 2002

Developments in recombinant DNA technology have provided an alternate route for the production of proteins.
Gene expression and production of proteins of interest are of great importance for basic research as well as for
biomedical applications. A number of expression systems using mammalian cells, insect cells, yeast and other
bacteria as host have been developed. Yeast has received attention as a suitable host for expression of many
mammalian genes due to many specific characteristics. Yeast strains, Schizosaccharomyces pombe, Saccharomyces
cerevisiae and Pichia pastoris, have many advantages over other systems and may be the host of choice for the
expression of complex proteins of therapeutic value. During the post-genomicera, the importance of these strains for
the expression of heterologous genes may enhance considerably.
Keywords: Schizosaceharomyces

pombe, Pichia pastoris, gene expression, glycosylation,secretion

Introduction
Antibodies have been used to identify, localize,
quantitate and analyze proteins. Before gene cloning,
only the natural sources were available for the
isolation of proteins, to which antibodies could be
raised. Low protein yields and the associated
impurities often compromised the quality and quantity
of antibodies.
However,
recent
advances
in
recombinant DNA technology have provided an
alternate route for the production of many proteins of
biomedical importance. The expression of foreign
genes and production of proteins of interest are very
important for both the basic research such as
elucidation of physiological activity, its modulation,
analysis of structure-function relationship of control
elements and regulation of gene expression as well as
practical applications
related to production of
pharmaceuticals. The demand for expression systems
suitable for high-level synthesis of foreign gene
products is, therefore, increasing rapidly.
The expression systems consist combinations of
various genetic elements of host and vector. A
number
of expression
systems,
some using
prokaryotes like Escherichia coli, Bacillus spp.,
Streptomyces
spp. as host and others using
Aspergillus, yeast, insect cells, mammalian cells and
other eukaryotes, have been developed (Shatzman,
* Author for correspondence:
Tel: 91-11-26089688; Fax: 91-11-26088874
E-mail: sk608@rediffmail.com

1993). In general, the expression of mammalian genes

using prokaryotes as host may sometimes result in an
inactive product due to incorrect folding or lack of
certain post-translational modifications, though the
manipulation of bacteria is easy and the production
cost is relatively low. In contrast, most of these
problems can easily be solved by expression of genes
using animal cells as host. However,
their
manipulation is not easy, the production levels are
low and the cost is high. Moreover, the mammalian
cell expression systems sometimes have the problem
of viral infection. More suitable expression systems
are thus desired, even though a number of expression
systems have already been developed. In present
review, current status of the characteristics and
importance of the currently available yeast cell
expression systems is summarized.
Yeast Expression System
Saccharomyces cerevisiae has been used in brewing
and bakery and is regarded as a safe organism based
on the genetics, molecular biology and physiology of
this traditional species (Romanos et ai, 1992). Yeasts
are lower eukaryotes present in both haploid and
diploid forms. Similar to other eukaryotic organisms,
the cell cycle of yeast is divided into G 1, S, G2 and M
phases and many biochemical functions of cell cycles
have been defined using a number of (cell cycle
defect) mutants (Strathern et ai, 1981). S. cerevisiae is
budding yeast as it can reproduce asexually by
asymmetrical division of cytoplasm. A haploid cell of

478

INDIAN J BIOTECHNOL,

S. cerevtsiae contains approximately 14,000 kb of

DNA, which exceeds the DNA content of E. coli by a
factor of 4. Apart from chromosomal DNA, which
constitutes approximately
90% of S. cerevisiae
genome, there are at least two other independent
genetic elements: the mitochondrial DNA and the 2f.l
plasmid. Some strains contain a third independent
replicon, the killer plasmid. These plasmids are
double stranded DNA coding for a toxin, which kills
other susceptible yeast strains (Wickner et al, 1981).
Both haploid and diploid cells are stable and
dominant as well as recessive mutants can be isolated
easily.
Yeasts have also received attention as a host for the
expression of animal proteins because: (i) Yeasts, like
bacteria, are single celled but unlike bacteria they are
eukaryotic and, therefore, the preferred organisms for
the expression of functional eukaryotic gene products;
(ii) They are simple to cultivate on inexpensive
growth media; (iii) Yeast strains are genetically well
characterized, detailed genetic maps are available for
S. cerevisiae and Schizosaccharomyces pombe (Petes,
1980); (iv) The molecular biology of yeasts is well
understood; (v) Their manipulation is easy; (vi)
Recombinants
can
easily
be
selected
by
complementation;
(vii) They have been used in
fermentation and brewery industry for a very long
time; and (viii) Their safety such as being free of
endotoxins has been guaranteed and so classified as
GRAS (generally recognized as safe) thus requiring
minimal toxicological studies.
General Yeast Vectors
The expression of proteins in yeast is often
undertaken for study of fundamental processes. Many
investigations require ectopic expression of a protein
under the control of promoters directing different
levels of expression. A convenient set of vectors has
been developed that allows the constitutive level of
foreign protein to be expressed over a range of three
orders of magnitude (Gatzke et al, 1995; Gilbert et al,
1994).
The list of plasmid vectors with different genetic
markers capable of transforming auxotrophic yeast
expression for a variety of cloning purposes has
greatly expanded.
By including
a gene that
complements one or more defective genes in the host
auxotroph within the plasmid expression cassette, the
recombinants can easily be detected on minimal
media. The usual strains have up to 6 different
selectable
markers,
HIS3
(imidazole
glycerol

OCTOBER 2003

phosphate dehydrogenase),
URA3 (orotidine 5'phosphate
decarboxylase),
TRP5
(tryptophan
synthetase),
LEU2
(p
isopropyl
malate
dehydrogenase), ARG2 (arginosuccinate lyase) and
CANI (canavanine). CANI confers sensitivity to
canavanine, an arginine analog that gets incorporated
in proteins, and is lethal to cells. It is an arginine
permease that allows canavanine to enter into host
cells. The CANI containing vectors can be used in
plasmid shuffling experiments where one version of a
gene, usually the wild type (WT), is replaced by
another version (the mutant) by selecting for the loss
of the WT vector on canavanine. This technique is
required if the original gene is an essential gene and
the cell cannot survive without it. A similar strategy is
followed with URA3 and a drug called 5-FOA
(fluroorotic acid) is used for this purpose. FOA is
converted into a toxin by URA3 and the presence of
URA3 on plasmid is lethal in the presence of FOA. In
this way, a URA3 plasmid can be cloned out of yeast.
FOA is very expensive and is used on very small
plates (Boeke et al, 1984). Following general category
of the yeast vectors are employed for different
purposes:
Integrative vectors. Yeast integrative plasmids
(YIps) consist of bacterial vector components and a
yeast gene with selectable marker. These cannot
survive in yeast as free plasmid as they lack an origin
of replication and a centromere.
Site-specific
integration of plasmid into host genome is mediated
by homologous recombination between chromosomal
DNA and vector DNA (Grallert et al, 1993). These
plasmids are used to carry foreign genes into the yeast
genome so that the selection pressure does not have to
be maintained and the gene will be expressed as a
single copy gene. Two different strategies have been
developed to integrate exogenous DNA into the yeast
chromosomes. The first approach involves the use of
YIps that lack an origin for autonomous replication
but carry sequences, which allow their integration to
chromosomes at high frequency. These plasmids are
linearized by a single restriction cut within the
complementary
yeast gene on the vector for
integrative gene conversion. A number of integrating
vectors have been used successfully to express a
variety of heterologous proteins in yeast. YIps with
two different yeast DNA sequences, one coding for
functional URA3 locus and the other coding for a
mutated "his3" gene, which is to be integrated into its
normal chromosomal site, have been constructed.
These contain two regions homologous to yeast

NASSER et al: YEAST EXPRESSION SYSTEM

genome and allow integration of foreign gene into

chromosomal DNA (Scherer & Davis, 1979).
The second approach for the gene integration is
gene replacement by homologous recombination. It is
the ability of complementary sequences, which align
and exchange the desired fragments in a double
crossover event. There is exact base-to-base exchange
in this process with no stop in the joints. The
frequency of homologous recombination is much
greater in yeast than in higher eukaryotes. Therefore,
it has been exploited as one of the most important
tools in the yeast genetics. This approach has also
been used for the elimination of yeast genes that could
interfere with efficient expression of desired foreign
protein. The transformation efficiency is very low
(one transformant/ug DNAII 07 _108 cells).

479
8gl11

Eco

Hind III

RI (4361)

Barn HI (375)

Sal (651)

1 Eco RI
2 Poll, dATP, dTIP/I
3 Ligase
(J
8gl11 Hind III

1 Hind III/Barn HI\

Replicating
vectors. Yeast replicating vectors
(YRps) contain prokaryotic plasmid DNA sequences
with part of a yeast DNA derived from YIp vectors
and also include chromosomal origin of replication.
The transformation efficiency of these plasmids is
2-3 fold higher than the YIp vectors. This high
frequency is thought to be due to presence of origin of
replication, which allows these vectors to replicate
autonomously. Such vectors are often referred as ARS
(autonomous
replicating
sequences)
vectors
(Stinchomb et al, 1979; Wohlgemuth & Bulboca,
1994). A prototype vector, YRp7 (Fig. 1), is 5.7 kb in
length (Stinchomb et al, 1979; Hitzeman et al, 1981).
However, often a rapid loss of these plasmids is seen
in growing cell population. Providing centromeric
(CEN) DNA sequences to YRp plasmids can solve
this problem. These sequences contain a central AT
rich region of 90 bp flanked by highly conserved
region of 11 and 14 bp respectively (Hieter et al,
1985; Caddle & Calos, 1994; Murkami et al, 1996).
Episomal vectors. Yeast episomal vectors (YEps)
contain prokaryotic sequences, a selectable yeast
marker gene and the entire 2 ~ plasmid also known as
"scp" plasmid. The 2 ~ plasmid is found in almost all
strains of S. cerevisiae in 50-100 copies per haploid
cell amounting to 2-3% of total chromosomal DNA
(Grimm et al, 1988; Smerdon et al, 1998). The most
striking structural feature of 2 ~ plasmid is the
presence of two inverted repeats of 599 bp which
divide the plasmid molecule into two non-identical
region. A recombination at these repeats yields two
forms (A and B) of this plasmid (Fig. 2). The 2 ~
plasmid possesses a single origin of replication and
replicates autonomously (Harteley & Donelson, 1980;
Smerdon et al, 1998).

2 Isolation of large
fragment
8gl11 Hind III

Barn HI (375)

Sal (651)

Fig. I-Construction

of Yrp7 vector and its derivative pFRL4

(Hitzeman et al, 1981)

Eco RI
(2407)
Hpa I

Form A

(2964)

Eco RI A 6318 - 2407

Eco RI B = 2407 bp

= 3912

bp

EcoRI
(2407)
Hpa I
(2964)

Form B
Eco RI A
Eco RI B

= 2407
= 4312

- 341 + 2006
- 2407 + 341

= 4072
= 2246

bp
bp

Fig. 2-Forms
A and B of the 2~ plasmid. The parallel lines
indicate the inverted repeats. Open bars represent the location of
the origin of replication. The locations of restriction sites have
been indicated, numbers represent the positions in form A
(Hartley & Donelson, 1980).

480

INDIAN J BIOTECHNOL,

Artificial
chromosome.
Normal
eukaryotic
chromosomes are always linear. The plasmids, when
supplied with ARS, the CEN sequences and a
functional telomere, can get replicated in autonomous
manner and are stably maintained in linear form as an
extra artificial mini-chromosome (Murray & Szostak,
1986). While highly stable, these vectors are not
useful for expression of foreign proteins in yeast.

Expression System Using Yeast as a Host

S. cerevisiae, the first species of yeast to be
employed for the production of recombinant proteins,
has been used as conventional host for protein
production for research, industrial and medical uses
(Romanos, 1995). Pichia pastoris and Hansela
polymorpha,
both methylotrophic
yeasts, were
originally developed for the large-scale and high-yield
production of heterologous proteins in a medium
containing methanol (Cregg et al, 1993; Bretthauer &
Castellino 1999). Other commercial yeast strains,
Kluyveromyces
lactis, Yarrowia lipolytica
and
Candida utilitis (Fleer et al, 1991; Kondo et al, 1995),
are phylogenetically closer to S. cerevisiae than
Schizosaccharomyces pombe. S. pombe is a promising
host for an expression system as it often provides
foreign gene products that are closer to their natural
form (Kaufer et al, 1985). Wild type yeasts are
prototrophic i.e. they are nutritionally self-sufficient
and capable of growing on minimal media.
Auxotrophic yeast strains, created using classical
genetics,
provide
the basis for selection of
successfully transformed strains. By including a gene
in the plasmid expression cassette, that complements
one or more defective genes in the host auxotroph,
one can easily select the recombinants on minimal
media. Hence, strains requiring leucine (Leu-2) will
grow on minimal media if they harbour a plasmid
expressing the Leu-2 gene. The number and variety of
S. cerevisiae and S. pombe strains possessing
nutritional markers make them attractive host, while
they have some limitations also.
Methylotrophic Yeast
The methylotrophic yeasts, H. polymorpha and P.
pastoris, can grow by utilizing methanol as the sole
source of carbon and are fast becoming the favourite
yeast hosts for expression of cloned genes. P. pastoris
has received widespread attention as an expression
system for its ability to express high level of
heterologous proteins (Sreekrishna et al, 1997;
Higgins & Cregg, 1998; Cregg, 1999). Pichia can
easily be cultured, genetically manipulated and has a

OCTOBER 2003

secretary pathway very similar to mammalian cells.

Both N-linked and a-linked glycosylation can take
place. The complex glycoproteins, abundant with Nlinked sites, expressed in Pichia are usually
biologically active. It is possible to engineer the
glycosylation
pathway
in Pichia
to obtain
glycoproteins very similar to those expressed in
mammalian cells (Bretthauer & Castellino, 1999). Of
more than120 heterologous proteins expressed in P.
pastoris, most are of human and other mammalian
origin (Cregg, 1999). P. pastoris has two advantages
over S. cerevisiae. Firstly, its methanol-inducible
alcohol oxidasel gene (AOXl) is tightly regulated,
repressed in absence of methanol and induced if
methanol is present (Tschopp et al, 1987). This can
conveniently be used to drive production of toxic
heterologous proteins, as it will not have any toxic
effect of heterologous protein to host until the
expression of the gene is induced by methanol
(Cereghino & Cregg, 2000). The second advantage is
that Pichia can be grown to very high densities up to
(100 g/l dry wt), which is hard to achieve with S.
cerevisiae (Cregg et ai, 1993). Pichia is particularly
advantageous for the production of therapeutically
relevant macromolecules in large amounts (Table 1)
(Pichuantes et al, 1996; Hollenberg & Gellisen, 1997;
Higgins & Cregg, 1998; Fischer et al, 1999;
Cereghino & Cregg, 2000).
Prototrophic Yeast
S. pombe, a unicellular eukaryote belonging to the
ascomycests family, is called fission yeast as it
reproduces by fission, besides through spores. Unlike
S. cerevisiae, no budding is observed in it. S. pombe is
the most intensively studied and well characterized
Table I-Expression

of heterologous proteines in Pichia pastoris

Protein

Reference( s)

I3r Adrenergic receptor

Bile salt-stimulated lipase
Caspase-3
Cathepsin V
CD38
Chorionic gonadotropin a-subunit,
l3-subunit and al3-heterodimer
Insulin
Insulin like growth factor-I (IGF-l)
Leukemia inhibitory factor (LIF)
Lymphocyte surface antigen CD38
Lysosomal u-manncsidase
Mast cell tryptase
Serum albumin
Tumour necrosis factor (TNF) a
Cereghino & Cregg, 2000

Weiss et al, 1998

Sahasrabudhe et al, 1998
Sun et al, 1997
Bromme et al, 1999
Munshi et al, 1997
Sen Gupta and Dighe, 1999
Kjeldsen et al, 1999
Brierley, 1998
Zhang et al, 1997
Fryxell et al, 1995
Liao et al, 1996
Chan et al, 1999
Ohtani et al, 1998 a.b
Sreekrishna et al, 1989

NASSER et al: YEAST EXPRESSION SYSTEM

yeast species than S. cerevisiae in terms of molecular

genetics and cell biology (Beach & Nurse, 1981;
Russel, 1989). However, unlike other yeasts, S.
pombe has many characteristics similar to higher
eukaryotic cells and it has not been used much
industrially to make wine, beer and bread. It is
gradually
being
considered
as very
useful
experimental model for the study of molecular
biology of yeast. Some mammalian genes can be
isolated using S. pombe by complementation of the
mutant homologue. The functional substitution of
human homologue of cell cycle regulator cdc2 for the
S. pombe cdc2 gene (which is homologous to the S.
cerevisiae CDC28 gene) is possible (Lee & Nurse,
1987). The similarity of human cdc2 system and that
of S. pombe has been confirmed at the protein level.
Some mammalian promoters are functional in S.
pombe
(Toyama
& Okayama,
1990). Higher
eukaryotic genes containing introns when introduced
into S. cerevisiae are not expressed, whereas the same
genes can be expressed in S. pombe (Kaufer et al,
1985). RNA splicing mechanism of S. pombe has
more similarity with higher eukaryotes than with S.
cerevisiae (Porter et al, 1990). A signal transduction
system of S. pombe shows marked similarities to
mammalian G-protein-coupled
system (Xu et al,
1994). The carbohydrate chains of yeast glycoproteins
are composed of N-linked and O-linked oligosaccharides with similar structure as in mammalian cells.
However, generally the glycans of yeast have outer
chains consisting of mainly mannose oligomers in Nlinked and O-linked glycosylation in contrast to the
glycoproteins derived from mammalian cells. S.
pombe, unlike other yeast species, has galactose
residues in mannose-rich sugar chains (Moreno et al,
1990; Ballou et al, 1994). It can be considered unique
yeast with characteristics
closer to those of
mammalian cells making it an accurate model for
molecular biology studies.
Table 2-Expression

481

Yeast Expression Vectors

For the construction of an expression system, after
deciding the host, an effectively
functioning
expression vector containing necessary elements for
the selected host is constructed to suit high-level
expression of the foreign gene. S. pombe molecular
genetics has led to greater understanding of the
components of each expression vector (Tohda et al,
1994).
Chromosomal Vectors
In these vectors, a foreign gene is stably maintained
within a chromosome in the host genome. Some
integrating vectors currently available for use in S.
pombe are based on complementation of S. pombe
mutations with S. cerevisiae genes including the leul
and ura4 (Grimm et al, 1988) or on the integration of
5 S ribosomal RNA gene (Smerdon et ai, 1998). The
analysis of genomic DNA has shown that the
transformants have one or more copies of the gene of
interest integrated into the host genome by
homologous recombination (Grallet et al, 1993).
Episomal Vectors
These vectors have the yeast origin of replication
and replicate extra-chromosomally in an autonomous
manner (Maundrell et al, 1988). In S. pombe
expression system, episomal vectors are commonly
used (Table 2). Following are the important
components of these vectors:
i) Origin of replication. It is essential for DNA
replication.
For autonomous
episomal
vectors
replicating outside the chromosomes, the 2 !l ori or
ars l are commonly used (Losson & Lacroute, 1983).
The 2 !l ori is the origin of replication of the 2 !l
plasmid of the S. cerevisiae. The arsl, a region of
chromosomal DNA of S. pombe, was identified as one
of the autonomously replicating sequences (ARSs)
and it promotes the extra-chromosomal autonomous

vector systems in Shizosaccharomyces

pombe

Vector

Origin of replication

Promoter

Yeast marker

pART
pEVP II
pCHY21
pREP
pTL2M UpAL 7
pSL2M UpAL 7
pSLF
pSLF 101
pSM 112

ars I
2J..lplasmid ori
ars I
ars I
ars II stb
ars II stb
ars I
ars I
2J..lplasmid ori

adh promoter
adh promoter
fbp I promoter
nmt I promoter
hCMV promoter
hCMV promoter
CaMV promoter
CaMV-tet
SV 40 promoter

LEU 2
LEU 2
URA 3
LEU 2
neol LEU 21 URA 3
neo/ LEU 21 URA 3
LEU 2
LEU 2
LEU 2

pART IIN795

ars I

adh promoter

LEU 2

CaMV, cauliflower mosaic virus; nea, neomycin resistance; tet, tetracyclin resistance gene Giga-Hama & Kumagai, 1999

482

INDIAN J BIOTECHNOL,

replication and maintenance of the plasmid outside

chromosomes (Wright et al, 1986). Plasmids having
S. pombe arsl are present in multiple copies (15-80),
however, these are mitotically unstable. Inclusion of
stb sequences (stabilizing sequence) derived from S.
pombe yield stable transformants both mitotically and
meiotically. When stb is combined with arsl, the
resulting vector can be maintained more stably in S.
pombe as a multiple copy vectors (up to 80
copies/cell). The loss frequency of this plasmid is
only about 13% per generation without the selection
pressure (Wright et al, 1986). The ARS elements in
transformants have been identified and have been
shown to co-localize with the chromosomal origin
(CaddIe & Calos, 1994).
ii} Selection markers. The most extensively used
protocols for transformation
of S. pombe are
electoporation (Hood & Stachow, 1990), protoplast
fusion (Beach & Nurse, 1981) and lithium acetate
mediated uptake of plasmid DNA (Okazaki et al,
1990). The lithium acetate method gives higher
frequency of transformation than the protoplast fusion
procedure. After transformation, the most commonly
used markers are LEU2 and URA3 genes of S.
cerevisiae, which complement the S. pombe mutants,
leul and ura4, respectively. Besides leul and ura4,
ade6, his3I and his7I are also used as selectable
markers (Burke, 1994; Waddell & Jerkins, 1995;
Cottarel, 1995). Although the sensitivity of S. pombe
to various antibiotics is low, the arninoglycoside
antibiotics
G418, hygromycin,
chloramphenicol,
pleomycin and bleomycin can conveniently be used as
selection markers (Burland et al. 1991). For selectable
markers, genes have been described for molecular
genetic manipulation of P. pastoris. Existing markers
are limited to the biosynthesis genes HIS 4 either from
P. pastoris or S. cerevisiae and ARG4 from S.
cerevisiae (Cregg & Madden, 1989; Cereghino &
Cregg, 2000).
iii) Promoter. Selection of a strong promoter is
critical for efficient
expression.
While many
promoters, including the promoters for the enzymes
of glycolytic pathway are used in S. cerevisiae
systems, only limited numbers of promoters are
known to work efficiently in S. pombe. Successful
expression of foreign genes in S. pombe has been
reported under the control of promoters for S.
cerevisiae such as PGK55, ADHI and CYC (Belsham
et al, 1986; Broker et al, 1987). A general expression
vector, pMB332 has been constructed for S. pombe
where the heterologous gene expression is driven by

OCTOBER 2003

adh promoter. The vector carries the URA3 gene of S.

cerevisiae, which complements the S. pombe ura4
mutation. The functional importance of this vector
system has been shown by the production of human
blood coagulation protein factor XIIIa (Broker &
Baumal 1989). One of the efficient promoters of
glycolytic enzymes in S. pombe is the promoter of
adhI gene, coding for alcohol dehydrogenase.
Vectors, pART and pEVPll, containing this promoter
are widely used (Broker et al, 1987). Promoters
derived from mammalian cells work efficiently with
S. pombe. They include SV40 promoter (Jones et ai,
1988), human cytomegalovirus (hCMV) promoter,
human chorionic gonadotropin a-subunit promoter
(Toyama & Okayama, 1990), adenovirus region 3
promoter (Swaminathan et al, 1993), HIV-l long
terminal repeat promoter (Toyama et al, 1992), and
human serum albumin TATA elements (Prentice &
Kingstan, 1992). In addition, cauliflower mosaic virus
(CaMV) 35S promoter and tomato nitrate reductase
(nia gene) promoter have also been reported to
function in S. pombe (Pobjecky et al, 1990; Truong et
al, 1992). A number of inducible promoters in S.
pombe, can be used effectively for the production of
toxic proteins. The most widely used inducible
promoter is nmtl promoter and an inducible vector,
pREP, has been developed utilizing this promoter
(Maundrell, 1990). The activity of this promoter is
suppressed by thiamin. A system, regulated by
tetracycline, combines the CaMV 35S promoter and
tetracycline regulatory region of plasmid (Faryar &
Gatz, 1992). Inducible promoters regulated by
glucose concentration include the fbp l promoter
(Hoffman & Winsten, 1989) and another promoter for
invl (Tanaka et al, 1998) coding for invertase in S.
pombe. Foreign genes, including GFP (green
fluorescent protein), have been expressed successfully
utilizing these promoters (Tanaka et al, 1998). GigaHama (1997) constructed a high-level expression
vector (Fig. 3a), which is used along with a
transducing vector (Fig. 3b) containing arsl/stb and
LEU21URA3 genes. It is believed that following cotransformation homologous recombination between
these vectors takes place inside the cell and the two
vectors replicate as a single plasmid.

Factors Involved in Homologous Expression

Yeasts are able to utilize many of the well-known
techniques used for the molecular and genetic
manipulation of growth characteristics in prokaryotes.
Yeast also has the capability of complex posttranslational modification. For the generation of

NASSER et al: YEAST EXPRESSION SYSTEM

SV40-T

stb

LEU2NRA3

Fig. 3-Structure
of the expression vectors. A. High-level
expression
vector pTL2M,
having hCMV promoter.
B.
Transducing vector pAL7/pAUS. HCMV-P, hCMV promoter,
SV40-P, SV40 promoter; SV40-T, SV 40 terminato; neo,
neomycin resistance gene; UTR, untranslated region; MCS,
multiple-cloning site; amp, ampicillin resistance gene; ori, origin
of replication; LEU21URA3, selection marker (Giga-Hama, 1997).

authentic recombinant proteins, following are the

important events for the stability of heterologous
expression:
Post-translational Modifications
The post-translational
targeting and membrane
translocation pathways have also been demonstrated
in S. cerevisiae (Rapoport et al, 1996a & b). The
cytoplasmic chaperone hsp 70 and its co-chaperones
maintain the translocation competency of protein
substrates, although they do not possess targeting
function. Genetically identified components have also
been
functionally
assessed
using
in
vitro
reconstitution systems. The studies demonstrated that
a heptameric membrane complex (including two
subcomplexes)
is essential for post-translational
processes (Panzner et al, 1995). One subcomplex
consists of four proteins (See 62p, See 63p, See 72p,
and See 73p), while other subcomplex, See 61p, is a
trimeric complex of See 61p, Sbh IP and Ssslp.
In contrast to the modification of the DNA
sequence,
each
alteration
introduced
at the
translational or post-translational level will be limited
to only one molecule and will not be further
amplified. This can take place in a heterogeneous
mixture of polypeptides. However, proteins with
differing properties can only be readily detected, if
present in large amounts. Codon usage can playa key
role in regulating gene expression and in the
production
of large quantities
of high-quality
heterologous protein. At the translational level, errors
can occur due to missense insertions by tRNAs whose
anticodons match two out of three codon bases.
Mistranslation can also occur if the mRNA from a
highly expressed heterologous gene contains rare
codons, or if the amino acid distribution were.
inordinately skewed relative to typical yeast protein". ':

483

The latter two possibilities can lead to ribosomal

pausing and frame shifting, thus reducing quantity or
quality of the desired gene product. Thus,
optimization of the codons in a cloned gene to fit the
preferences
of the yeast
cell
can
reduce
mistranslation, although this is relatively simple for
small proteins, it becomes more complex for larger
proteins.
Sometimes authenticity of a mature protein may
represent a more important consideration than the
highest level of expression specially if the biological
activity and physiochemical
properties such as
solubility, biodistribution, circulatory half-life or
stability are dependent on specific post-translational
modifications. This is the case when heterologous
genes are expressed in cells that lack the appropriate
modification system, or if the production of a
heterologous protein exceeds the post-translational
capacity of the host cell. Many post-translational
modifications of proteins are correctly done in yeast
cells.
Intracellular Expression
Recombinant proteins have been produced in yeast
both in intracellular and secretion expression systems.
Intracellular expression is liked for heterologous
proteins, which are normally expressed in the
cytoplasm, as well as for those secreted proteins that
have no or relatively few disulphide bonds. Yeast can
carry out the post-translational removal of the initiator
methionine by the yeast methiony I aminopeptidase
from the cytoplasmically expressed proteins. Where
the amino-terminal methionine residue is retained,
problems of immunogenicity
may arise during
medical applications. In addition to the removal of the
amino-terminal methionine, yeast also carries out
amino-terminal
acetylation,
carboxy-terminal
methylation, myristylation and farnesylation. The
latter three are important in the membrane targeting of
intracellularly expressed proteins. Yeasts are also
capable of assembling intracellularly
expressed
oligomeric proteins such as hepatitis B surface
antigen (Valenzuala et al, 1982; Cregg et at, 1987;
Janowicz et al, 1991) and the subunits of mammalian
Na+, K+-ATPase (Eakle et al, 1992; Horowitz et al,
1990). Hoffman et al (1.995) reported the high-level
expression and functional assembly of the three
human embryonic hemoglobins, Gower I (2 2),
Gower II (2 2) and Portland (2 2) by S. cerevisiae.
Each of these proteins is a functional tetramer of the
general form, a2b2. The different chains were
correctly processed at their amino termini and four

484

INDIAN J BIOTECHNOL,

OCTOBER 2003

heme groups bound to the protein imitated the native

protein. An acetyl group blocks the amino-terminal
amino acid of the recombinant chain, as is the case in
the naturally occurring molecule. The expression and
processing of the hepatitis C virus core protein
(HCcAg) were analyzed in methalotropic yeast, P.
pastoris. The proteolytic processing of the precursor
in P. pastoris produced two proteins (21 kDa and 23
kDa), detected as the major product of HccAg, which
have same N-terminal end and both react with a
monoclonal antibody against the first 35 amino acids
of HCcAg. The correct proteolytic processing of the
recombinant protein proves the usefulness of P.
pastoris as an expression system to understand the
processing of HCV structural proteins (Acosta-Rivero
et al, 2002).

modifications for its maturation to biologically active

form. S. cervisiae was chosen as a model to study the
events related to endoproteolytic processing of proCCK. Expression of pro-CCK as a fusion protein to
the pre-pro leader peptide of a-mating factor directed
the protein through the secretary pathway and resulted
in secretion of CCK with a glycine extension
(Johnson et al, 2001). Higher levels of expression of
the G-coupled human dopamine and mouse 5-HT5A
serotonin receptors has been achieved in S. pombe
(Sander et al, 1994) and P. pastoris (Weiss et al,
1998) than in S. cerevisiae. The surface expression of
foreign proteins in yeast is generating substantial
interest in applications such as biocatalysis, wholecell vaccines, and combinatorial library presentation
(Schreuder et al, 1996).

The ability of yeast to perform various posttranslational modifications and targeting of proteins to
specific cell membranes has led to its evaluation as an
expression system for membrane proteins (Evans et
al, 1995; Grisshammer & Tate, 1995). Membrane
proteins
require
complex
post-translational
modifications
including
interaction
with other
proteins, such as molecular chaperones, to attain their
final conformation and insertion into membranes. Due
to the poor understanding of the factors that are
important for membrane protein insertion and folding,
there are still only a few examples of highly expressed
heterologous membrane proteins in yeast. Often, the
initial expression experiments are performed in a host
that is homologous to the source of the membrane
protein. Plant and fungal membrane proteins are more
readily expressed in S. cerevisiae than the mammalian
membrane proteins (Villalba et al, 1992; Reutz &
Gros, 1994; Grisshammer & Tate, 1995). However,
there are some reports on expression of human
membrane proteins, including the erythroid anion
exchanger AEI (Sekler et al. 1995), the emopamilbinding protein (Hanner et al, 1995), the multiple
drug resistance related P-glycoprotein (Evans et al,
1995), the f3-adrenergic receptor (Grisshammer &
Tate, 1995), the receptors for dopamine (Mak et al,
1994), transferrin, retinoid X (Mak et al, 1994) and
estrogen (Arnold et al, 1996), and the Neurospora
crassa plasma membrane H+-ATPase (Mahanty &
Searborrugh, 1996) by yeast. By using yeast system,
sufficient amounts of functional protein suitable for
biochemical,
pharmacological
and
biophysical
analysis
can
be
obtained.
The
vertebrate
neuroendocrine peptide, cholecystokinin (CCK), is
subjected
to a number
of post-translational

The undetectable levels of direct cytoplasmic

expression of an authentic protein are relatively
common problem. Yield and the biological activity of
a recombinant protein may be increased by fusing it to
a rapidly folding protein such as thioredoxin, which
facilitates its folding and enhances solubility. An
alternate approach may be to fuse it to a peptide tag.
which may be recognized by a commercially available
monoclonal antibody (Kaslow et al, 1994). In such
instances, the problem can be overcome by fusion of
the desired protein to stable proteins such as human
superoxide dismutase (hSOD) or human y-interferon
Cy-IFN). The hSOD fusion approach has been used to
overexpress a number of viral polypeptides for
diagnostic purposes (Kuo et al, 1989; Barr et ai,
1987). These yeast-expressed proteins have been
purified and incorporated into diagnostic kits for the
screening of blood for HIV -1 and hepatitis C virus
(HCV) antibodies. Often, the fusion strategy results in
an insoluble product that must be extracted and
subjected to an in vitro cleavage and refolding
process, which occurs with variable efficiency and
may be difficult for certain complex structures
resulting in low yield of a authentic product of highquality. The difficulty of refolding and generation of
recombinant proteins with authentic amino termini
may be overcome by their fusion to ubiquitin (Ub), a
yeast hydrolase, a 76 amino acid protein derived from
the processing of a larger precursors. Ub fusion
expression system takes advantage of the processing
of the Ub precursor in vivo by an endogenous yeast
hydrolase. The yeast Ub system, closely related to a
similar mammalian system, can be conveniently used
to express foreign proteins as fusion protein. When a
chimeric gene encoding an Ub:f3-galactosidase fusion

NASSER et al: YEAST EXPRESSION SYSTEM

protein was expressed in yeast, highly stable

expression was obtained. Ub was later cleaved from
the nascent fusion protein to obtain high yields of ~galactosidase (Finley et at, 1984). When making
products intracellularly, the Ub fusion approach has
two distinct advantages. First, it can significantly
enhance the yield and/or stability of proteins that are
otherwise unstable in the cytosol, the amino-terminal
Ub moiety of the fusion possibly prevents immediate
degradation of the fusion partner (Ecker et at, 1989;
Sabin et at, 1989). Secondly, apart from proline, it can
generate a protein with any desired amino terminus,
because
Ub hydrolase
processing
is largely
independent of the amino terminus of the protein
fused to Ub (Sabin et at, 1989).
Extracellular Expression
The secretion of heterologous proteins into the
culture medium offers a way to avoid toxicity from
accumulated material and simplify purification of the
protein, because the yeast organism secretes relatively
low levels of native proteins. Furthermore, the
passage of the proteins through the secretory pathway
permits post-translational events such as proteolytic
maturation,
glycosylation
and disulphide
bond
formation. The secretion of heterologous proteins is
driven by virtue of a cleavable, amino-terminal signal
sequence that is derived from either the native protein
or the leaders of the S. cerevisiae prepro-mating factor
or invertase. Numerous examples demonstrate the
capability of yeast to secrete mature human
polypeptides possessing the expected biological, and
in most cases, physico-chemical properties. Some
such examples include the production of single chain
Fv fragments (Luo et at, 1995; Ridder et at, 1995),
erythropoietin
receptor (Nair & Harris, 1995),
thrombomodulin (White et at, 1995), factor XII,
gastric lipase (Crabe et at, 1996; Smerdon et at,
1998), fibroblast collagenase (proMMPl) (Rosenfeld
et at, 1996), analogs of tissue factor pathway inhibitor
(Peterson et at, 1996), and interleukin-8 (WernetteHammond et at, 1996). In all these cases, specific
structure/function
relationships
such as catalytic
activity, ligand binding, ATP-dependent
efflux,
specific anti-idiotype binding and clot promotion
capacity were found to be comparable with the native
counterpart. It is not surprising that yeast secretes
non-human proteins, such as coffee bean ~galactosidase (Zhu et at, 1995), bovine enterokinase
(Vozza, 1996), and Schistosoma mansoni cathepsin B
(Lipps et at, 1996), that are equivalent to the native

485

molecule. Cathepsin B is a good example of a protein

that could not be expressed successfully in bacterial
or insect cell host systems, but was secreted in a
functional, authentic form by yeast.
Rational engineering can be used to produce novel
proteins. P. pastoris has been used to efficiently
express rabbit Sc FV antibody fragment that
recognizes human leukaemia inhibitory factor isolated
from a combinatorial library (Ridder et al, 1995).
However, the removal of a signal sequence by
specific signal peptidase may sometimes be defective
resulting in the production of a modified protein. It is
reported that the removal of the a-factor leader
sequences by the Kex2 protease (Kex2p) is
incomplete, resulting in hyperglycosylated secreted
material with an amino-terminal extensions. Kjeldsen
et at (1999) have shown that the introduction of an
amino-terminal spacer between the a-factor leader
and the insulin precursor significantly improved
Kex2p processing. The spacer peptide is then
removed either in vitro by a specific protease or ill
vivo by the yeast aspartyl protease 3. This
modification also increased fermenter yields of the
insulin precursor by 215%.

Protein Folding and Secretion

The secretion of properly folded proteins, crucial
for' correct biological activity, is one of the major
factors for considering yeast as the preferred host for
heterologous protein expression. This is because the
direct capture of active product from conditioned
medium eliminates the need for costly and lowyielding cell disruption or refold process steps.
Although there are some exceptions, most notably
human serum albumin, which is secreted at 4 gl/ 1 by
P. pastoris (Faber et at, 1995), the high productivity
expected from the high-copy number yeast vectors
containing foreign genes driven by very strong
promoters is usually not obtained for secreted
proteins. To better understand the mechanistic nature
of this problem, Parekh et at (1995) have been
studying bovine pancreatic trypsin inhibitor (BPTI) as
a model of secretion and the role of folding in the
endoplasmic reticulum (ER). In this system, the level
of expression driven by a multicopy (>50), 2/.l-based
plasmid vector was found equivalent to that driven by
a single copy construct. There was higher gene
transcription in the multicopy system. However, the
binding of conformationally
specific antibodies
showed that majority of the proteins in cells
harbouring the multicopy construct were improperly

INDIAN J BIOTECHNOL,

486

folded and were retained in lumen of the ER

(Robinson & Wittrup, 1995). This suggests that
maintenance of correct intralumenal tertiary structure
may be one of the major factors for limitation of
expression through the eukaryotic secretion pathway.
Many types of proteins could possibly be expressed
in heterologous systems. Secretory proteins, on
expression,
need to go through the reticuloendothelial system for post-translational processing.
The secretory signal, recognized by the signal
peptidase, is essential for passing through the correct
secretory pathway. Only a limited number of foreign
secretory proteins have been produced in S. pombe
(Table 3). In case of human antithrombin II, human
gastric lipase, human placental alkaline phosphatase
and S. cerevisiae invertase, the secretory signals of
the protein worked effectively in S. pombe, resulting
in the secretion of the proteins (Broker et al, 1987).
However, in case of many other secretory proteins,
signal peptide is not recognized by the signal
peptidase of S. pombe, so the precursor proteins do
not enter the endoplasmic reticulum but stay in the
cell cytoplasm without undergoing maturation.
High expression of granulocyte-colony stimulating
factor (G-CSF), erythropoietin (EPO) or S. pombe
acid phosphatase in S. cerevisiae leads to low levels
of extractable heavy chain binding protein (BiP) and
protein disulphide isomerase (PDI), which are key
proteins involved in lumenal folding. By showing that
BiP synthesis rate was not reduced as a result of GCSF expression and that augmented level of BiP
could not be restored by its co-expression, it was
generally suggested that foldase losses could be due
to degradation or aggregation (possibly as a result of
their own misfolding) (Robinson & Wittrup, 1995)
Unfortunately,
the exact mechanism by which
Table 3-Production

of heterologous proteins in S. pombe

Protein

Reference

Human lipocortin I
Human blood coagulation factor Xllla
B-Glucuronidase
HIV type-I protein R
Human Bradrenergic receptor
Human D2s dopamine receptor
Human P-glycoprotein
Human liver epoxide hydrolase
Cytochrome P450
Human gastric lipase
Human antithrombin III
Human interleukin-6
Human placental alkaline phosphatase
Giga-Harna & Kumagai, 1999

Giga-Hama et al, 1994

Broker & Baumi, 1989
Pobjecky et ai, 1990
Zhao et al, 1996
Ficca et al, 1995
Sander et al, 1994
Ueda et ai, 1993
Jackson & Burchell, 1988
Yasumori, 1997
Smerdon et ai, 1995
Broker et ai, 1987
Giga-Hama, 1997
Sambamurti, 1997

OCTOBER 2003

lumenal proteins regulate the folding and secretion is

not fully understood and the productivity potential of
yeast remains underexploited.
Glycosylation
It is both, the most common and the most complex
form of post-translational modification (Meyniataalles
& Combes, 1996). The majority of therapeutically
relevant proteins are usually glycoproteins in nature
and must therefore be properly glycosylated to display
the correct biological activity. Thus, the monitoring of
glycosylation
patterns
in quality
control
of
recombinant therapeutic proteins to assure product
safety, efficacy and consistency
is assurninz
importance. It is postulated that general function of
protein glycosylation is to aid in the foldinzb of
nascent polypeptide chain and in stabilization of the
conformation of mature glycoprotein (Wang et al,
1996). The nature of polypeptide, the host-cell
phenotype and the cell environment can determine the
glycosylation pattern and hence the quality of the
resulting glycoprotein.
Although a number of recombinant glycoproteins
of pharmaceutical or industrial value have been
obtained using yeast expression system, they often
have altered biological properties and functions as
compared to their native counterparts. This is mostly
due to differences in protein glycosylation. Alteration
in normal glycosylation patterns of therapeutic
proteins may affect their in vivo function in respect to
solubility, sensitivity to proteases, serum half-life, and
biological activities such as targeting to specific cells
or interaction with specific receptors. Yeast cells
recognize the N-glycosylation recognition sequence
similar to higher eukaryotic cells, indicating that they
have the potential to glycosylate at the same sites as
the
higher
eukaryotic
cells.
However,
the
carbohydrate
moieties
of yeast
glycoproteins
primarily consist of man nose residues appended in
different linkages to the core glycosyl units. The
recombinant glycoproteins generated in S. cerevisiae,
being high-rnannose type, will be recognized by the
man nose receptors on various cells and removed from
the circulation when injected into mammalian species.
In addition, non-human glycosylation patterns are
potentially
immunoreactive
to
humans.
The
hyperglycosylation very often dilutes the advantages
that the microbial eukaryote S. cerevisiae might have
over E. coli or mammalian cell expression system.
Mannan mutants, which exhibit less elaborate Nlinked glycosylation, have been isolated. These
.

NASSER et al: YEAST EXPRESSION SYSTEM

mutants as well as other yeast strains, however, do not

grow. Certain yeast species such as P. pastoris and H.
polymorpha
seem
to
be
less
prone
to
hyperglycosylating
the
heterologous
proteins
(Romanos et al, 1992; Stratton-Thomas et al, 1995).
The average length of mannose residues in proteins
produced in these two yeasts is only 8-14 monomers,
compared to 50-100 monomers in proteins produced
by S. cerevisiae (Giga-Hama & Hiromichi, 1999).
Due to this problem, often the mammalian cells are
the preferred host cells for the generation of
recombinant glycoproteins for therapeutics purposes.
However, all the steps involved in eukaryotic protein
trafficking and post-translational modification may
not be same in yeast and the higher eukaryotes
(Sadhukhan & Sen, 1996). This conclusion was drawn
from the work done on testicular isozymes of
angiotensin-converting
enzyme (ACET), where the
contributions of each of five potential N-glycosylation
sites of ACET toward its synthesis, glycosylation,
intracellular
transport,
cleavage
secretion
and
enzymatic activity were studied. The a-linked
glycans of S. cerevisiae also differ from those of
higher eukaryotes. Human urokinase plasminogen
activator (UPA) epidermal growth factor-like domain
is post-translationally modified by an unusual 0linked fucosy lation in both naturally isolated UP A
and recombinant UPA produced in mammalian
culture, but not in S. cerevisiae (Stratton-Thomas
et al, 1995).
Until recently, some questions
were raised
regarding the presence of a-linked glycans in Pichia
proteins. In general, such glycans are present but have
relatively shorter chain length than those found in
either S. cerevisiae or in mammalian system. These
differences may not always influence biological
activity of the protein, but influence certain in vivo
performances such as the half-life of the protein.
Though there are examples where differences in
glycosylation of r-proteins did not result in any
change in in vivo performances. It may be possible to
engineer the glycosylation pathway in P. pastoris to
obtain glyco-conjugates having similarity with the
structure of proteins expressed in mammalian cells.
There has been discussions about feasibility of having
such approaches for both mammalian and insect cells
(Hogland et al, 1998; Hironaka et al, 1992; Kumar et
al, 1996; Fusseneggar et al, 1999; Jarvis et al, 1999).
The ability to genetically engineer the yeast cells
and the increasing available knowledge of the host
machinery
necessary
to form
the complex

487

carbohydrate structures found on many mammalian

proteins provides the opportunity to specifically
optimize the host-cell background that will produce
proteins of pharmaceutical interest with the desired
carbohydrate structures. S. pombe gm 1+ gene encodes
an
UDP-galactose
transporter
for
protein
galactosylation (Tabuchi et al, 1997; Tanaka et 01,
2001). Further, mutant deficient in UDP-galactose
transport activity has been isolated (Takegawa et 01,
1996; Tanaka et al, 2001). However, because it is not
possible to make meaningful generalizations about the
optimal
glycosylation
pattern
of recombinant
therapeutics, each glycoprotein must be individually
assessed in the context of both the systems in which it
is expressed and the desired clinical benefit.
A certain degree of heterogeneity
within a
recombinant glycoprotein is permitted by regulatory
agencies
provided
that efficacy,
safety
and
consistency can be demonstrated. Glycosylation
consistency can also be monitored and comparative
studies of structure and function are becoming easier
with the development of automated and sensitive new
techniques (O'Neill, 1994; Garcia et al, 1995; Cregg,
1999).
Improving Expression of Foreign Proteins
Yeast can be grown as haploid, diploid or polyploid
cells. Cell size increases with increasing ploidy of the
chromosomes. Haploid cells are favoured in basic
research for genetic manipulation. However, use of
polyploid yeast is less likely to result in
spontaneously arising deleterious mutant phenotypes
over many generations of growth. Strain stability is an
important' consideration in fermentation technology,
since large-scale production is very sensitive to
alterations in genetic background of the host strain.
Stable auxotropic mutants are essential for the
selection of transformants in all yeast species (Cregg
et al, 1985; Dohmen et al, 1989). Deletions in the loci
for the auxotropic markers are preferred to point
mutations. The genetic background of the host yeast
strain can affect the production and/or recovery of the
heterologous proteins. The use of protease deficient
strains (Brierley et al, 1998; White et al, 1995) has
been crucial for the expression of a number of
authentic mammalian (Barr et al, 1987) and viral
(Bathurst et al, 1989) proteins. An additional protease
deficient strain, SMD 1168~ pep4:: URA3~ Kex 1::
SUC2 his4 ura3, inhibits proteolysis of murine and
human endostatin (Boehm et al, 1999). Mutants, that
confer resistance to the cloned genes, can be selected

488

INDIAN J BIOTECHNOL,

for cytotoxicity prevented efficient protein expression

such as with IGF-l. Isolation of a class of yeast super
secreting mutants that support high level secretion of
foreign proteins, have been achieved by mutagenesis
of yeast strains followed by the screening of mutants
for elevated protein secretion.

Generating Muiticopy Strains

Optimization of protein expression often includes
the isolation of multi copy expression strains, which
contain multiple integrated copies of an expression
cassette and sometimes yield more heterologous
protein than a single copy strain (Clare et al, 1991).
Three approaches led to multi copy expression strains
of P. pastoris.
First approach
involved the
construction of a vector with multiple head-to-tail
copies of an expression cassette (Brierley, 1998). A
particular advantage of this approach, especially in
the production of human pharmaceuticals, is that the
precise number of expression cassettes is known and
can be recovered for direct verification by DNA
sequencing.
Second method. utilizes expression
vectors containing the P. pastoris HIS 4 and bacterial
Tn903 Kan' genes. The bacterial
kanamycin
resistance gene also confers resistance to the related
eukaryotic antibiotic G418 (Scorer et al, 1994). This
method results in subset of colonies enriched for those
containing multiple vector copies. However, the
vector copy number varies greatly. Thus, a significant
number (50-100) of transformants must be subjected
to further analysis of copy number and expression
level. The third approach to construct the multicopy
strains involves the use of a vector with the bacterial
hie gene, which confers resistance to the antibiotic
zeocin (Higgins et al, 1998). Unlike G418 selection,
stains transformed with expression cassette containing
the zeocin marker can be selected directly by
resistance to the drug. Additionally, plating on
increased concentration of zeocin in selected plates
can enrich populations of the transformants for
multicopy expression cassette strains. Also, as the sh
hie gene can serve as selectable marker in both
bacteria and yeast, these expression vectors are
compact and convenient to use.
High Cell Density Growth in Fermenter Cultures
Yeasts have many advantages of both the
prokaryotic and the eukaryotic systems. They are
faster, easier and less expensive to grow, the process
can be scaled up and the level of protein expression is
very high (10-100 fold) as compared to that of E. coli

OCTOBER 2003

(Faber et al, 1995; Vozza et al, 1996). Further, the

protein can be expressed as secretary protein, which
helps in downstream processing and purification of
the gene product.
P. pastoris, a relatively poor fermenter, may be an
advantage during industrial use, as in high cell density
cultures of S. cerevisiae, the ethanol (the product of S.
cerevisiae fermentation) rapidly builds up to toxic
levels and hinders further growth of cell and the
foreign protein production. With its preference for
respiratory growth, P. pastoris can be cultured at
extremely high densities (500 A6oo1ml) under
controlled environments of fermenters with little risk
of 'pickling itself (Stratton et al, 1998). High-density
growth is especially important for secretary proteins,
as the concentration of product in the medium is
directly proportional to the concentration of cells in
the culture. Another positive aspect of growing P.
pastoris in fermenter cultures is that the level of
transcription initiated from the AOXI promoter can be
3-5 times greater in cells fed methanol at growth
limiting rates as compared to cells grown in excess of
methanol.
A continuous fermentation process has been
developed for P. pastoris expression system. P.
pastoris glyceraldehyde-3-phosphate
dehydrogenase
promoter has been used to produce large quantities of
recombinant human chitinase for pre-clinical studies
as a potential anti-fungal drug (Goodrick et al, 200 I).
Expression levels of about 200 to 400 mg/ml have
been demonstrated in fed-batch fermentation using
strains with either the traditional methanol-inducible
or the constitutive GAP promoter. Proteolytic
degradation- of the enzymes was typically seen in fed
batch fermentation. No proteolytic degradation of the
enzyme was seen in the continuous fermentation
mode.
A hall mark of the P. pastoris system is the ease
with which expression strains scale up from shake
flask to high density fermenter cultures. Although
some foreign proteins have expressed well in shake
flask cultures, expression levels are typically low
compared to fermenter cultures (Gleeson et al, 1998;
Clare et al, 1998). The growth conditions for P.
pastoris are ideal for large-scale production of
heterologous protein because the medium components
are in-expensive and defined, consisting of pure
carbon source (glycerol and methanol), biotin, salts,
trace elements and water. This medium is free of
undefined ingredients that can be sources of pyrogens
or toxins and is, therefore, convenient for the

NASSER et al: YEAST EXPRESSION SYSTEM

production of human pharmaceuticals. Also, as P.

pastoris is cultured in media with a relatively low pH
and methanol, it is less likely to become contaminated
with most other microorganisms.
Conclusions
Many protein types could possibly be expressed in
heterologous systems. Only a limited number of
foreign secretory proteins have been produced in S.
pombe. The use of S. pombe for foreign gene
expression has a big advantage, as it has a well
developed Golgi apparatus and the galactosyl
transferase enzyme system, that are not found in many
other yeasts.
Most
of the post-translational
modifications and correct maturation of expressed
proteins are, therefore, possible in S. pombe. The
genome sequence of S. pombe (Wood et al, 2002) will
provide further insights in the development of the new
expression strategies for the high level expression of
proteins. The development of new generation of
expression vectors has opened up a way for massproduction of proteins of biomedical importance in an
economic manner using S. pombe. Hopefully, the
additional expression vectors for S. pombe will be
developed.
However,
S. pombe
has
some
disadvantages such as the choice of the vectors is still
limited and the issue of biological activity of
expressed proteins is unresolved. It is expected that
these problem will soon be resolved and S. pombe
will become one of the host of choice for high-level
expression of many proteins.
Acknowledgement
The authors thank Mr A Mahesh and Mr P Gautam
for manuscript preparation. Financial assistance from
ICAR as an ad hoc research project to S K J is
gratefully acknowledged. M W N and V P are Senior
Research Fellows of CSIR and ICMR, respectively.
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