Evaluation of Yeast as an Expression Systemnopr.niscair.res.in/bitstream/123456789/11338/1/IJBT 2(4)...

Indian Journal of BiotechnologyVol 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 DNAtechnologyhave 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 forbiomedical applications. A number of expression systems using mammalian cells, insect cells, yeast and otherbacteria as host have been developed. Yeast has received attention as a suitable host for expression of manymammalian genes due to many specific characteristics. Yeast strains, Schizosaccharomyces pombe, Saccharomycescerevisiae and Pichia pastoris, have many advantages over other systems and may be the host of choice for theexpression of complex proteins of therapeutic value. During the post-genomicera, the importance of these strains forthe expression of heterologous genesmay enhance considerably.

Keywords: Schizosaceharomyces pombe, Pichia pastoris, gene expression,glycosylation,secretion

IntroductionAntibodies have been used to identify, localize,

quantitate and analyze proteins. Before gene cloning,only the natural sources were available for theisolation of proteins, to which antibodies could beraised. Low protein yields and the associatedimpurities often compromised the quality and quantityof antibodies. However, recent advances inrecombinant DNA technology have provided analternate route for the production of many proteins ofbiomedical importance. The expression of foreigngenes and production of proteins of interest are veryimportant for both the basic research such aselucidation of physiological activity, its modulation,analysis of structure-function relationship of controlelements and regulation of gene expression as well aspractical applications related to production ofpharmaceuticals. The demand for expression systemssuitable for high-level synthesis of foreign geneproducts is, therefore, increasing rapidly.

The expression systems consist combinations ofvarious genetic elements of host and vector. Anumber of expression systems, some usingprokaryotes like Escherichia coli, Bacillus spp.,Streptomyces spp. as host and others usingAspergillus, yeast, insect cells, mammalian cells andother eukaryotes, have been developed (Shatzman,

*Author for correspondence:Tel: 91-11-26089688; Fax: 91-11-26088874E-mail: [email protected]

1993). In general, the expression of mammalian genesusing prokaryotes as host may sometimes result in aninactive product due to incorrect folding or lack ofcertain post-translational modifications, though themanipulation of bacteria is easy and the productioncost is relatively low. In contrast, most of theseproblems can easily be solved by expression of genesusing animal cells as host. However, theirmanipulation is not easy, the production levels arelow and the cost is high. Moreover, the mammaliancell expression systems sometimes have the problemof viral infection. More suitable expression systemsare thus desired, even though a number of expressionsystems have already been developed. In presentreview, current status of the characteristics andimportance of the currently available yeast cellexpression systems is summarized.

Yeast Expression SystemSaccharomyces cerevisiae has been used in brewing

and bakery and is regarded as a safe organism basedon the genetics, molecular biology and physiology ofthis traditional species (Romanos et ai, 1992). Yeastsare lower eukaryotes present in both haploid anddiploid forms. Similar to other eukaryotic organisms,the cell cycle of yeast is divided into G 1, S, G2 and Mphases and many biochemical functions of cell cycleshave been defined using a number of (cell cycledefect) mutants (Strathern et ai, 1981). S. cerevisiae isbudding yeast as it can reproduce asexually byasymmetrical division of cytoplasm. A haploid cell of

478 INDIAN J BIOTECHNOL, OCTOBER 2003

S. cerevtsiae contains approximately 14,000 kb ofDNA, which exceeds the DNA content of E. coli by afactor of 4. Apart from chromosomal DNA, whichconstitutes approximately 90% of S. cerevisiaegenome, there are at least two other independentgenetic elements: the mitochondrial DNA and the 2f.lplasmid. Some strains contain a third independentreplicon, the killer plasmid. These plasmids aredouble stranded DNA coding for a toxin, which killsother susceptible yeast strains (Wickner et al, 1981).Both haploid and diploid cells are stable anddominant as well as recessive mutants can be isolatedeasily.

Yeasts have also received attention as a host for theexpression of animal proteins because: (i) Yeasts, likebacteria, are single celled but unlike bacteria they areeukaryotic and, therefore, the preferred organisms forthe expression of functional eukaryotic gene products;(ii) They are simple to cultivate on inexpensivegrowth media; (iii) Yeast strains are genetically wellcharacterized, detailed genetic maps are available forS. cerevisiae and Schizosaccharomyces pombe (Petes,1980); (iv) The molecular biology of yeasts is wellunderstood; (v) Their manipulation is easy; (vi)Recombinants can easily be selected bycomplementation; (vii) They have been used infermentation and brewery industry for a very longtime; and (viii) Their safety such as being free ofendotoxins has been guaranteed and so classified asGRAS (generally recognized as safe) thus requiringminimal toxicological studies.

General Yeast VectorsThe expression of proteins in yeast is often

undertaken for study of fundamental processes. Manyinvestigations require ectopic expression of a proteinunder the control of promoters directing differentlevels of expression. A convenient set of vectors hasbeen developed that allows the constitutive level offoreign protein to be expressed over a range of threeorders of magnitude (Gatzke et al, 1995; Gilbert et al,1994).

The list of plasmid vectors with different geneticmarkers capable of transforming auxotrophic yeastexpression for a variety of cloning purposes hasgreatly expanded. By including a gene thatcomplements one or more defective genes in the hostauxotroph within the plasmid expression cassette, therecombinants can easily be detected on minimalmedia. The usual strains have up to 6 differentselectable markers, HIS3 (imidazole glycerol

phosphate dehydrogenase), URA3 (orotidine 5'-phosphate decarboxylase), TRP5 (tryptophansynthetase), LEU2 (p isopropyl malatedehydrogenase), ARG2 (arginosuccinate lyase) andCANI (canavanine). CANI confers sensitivity tocanavanine, an arginine analog that gets incorporatedin proteins, and is lethal to cells. It is an argininepermease that allows canavanine to enter into hostcells. The CANI containing vectors can be used inplasmid shuffling experiments where one version of agene, usually the wild type (WT), is replaced byanother version (the mutant) by selecting for the lossof the WT vector on canavanine. This technique isrequired if the original gene is an essential gene andthe cell cannot survive without it. A similar strategy isfollowed with URA3 and a drug called 5-FOA(fluroorotic acid) is used for this purpose. FOA isconverted into a toxin by URA3 and the presence ofURA3 on plasmid is lethal in the presence of FOA. Inthis way, a URA3 plasmid can be cloned out of yeast.FOA is very expensive and is used on very smallplates (Boeke et al, 1984). Following general categoryof the yeast vectors are employed for differentpurposes:

Integrative vectors. Yeast integrative plasmids(YIps) consist of bacterial vector components and ayeast gene with selectable marker. These cannotsurvive in yeast as free plasmid as they lack an originof replication and a centromere. Site-specificintegration of plasmid into host genome is mediatedby homologous recombination between chromosomalDNA and vector DNA (Grallert et al, 1993). Theseplasmids are used to carry foreign genes into the yeastgenome so that the selection pressure does not have tobe maintained and the gene will be expressed as asingle copy gene. Two different strategies have beendeveloped to integrate exogenous DNA into the yeastchromosomes. The first approach involves the use ofYIps that lack an origin for autonomous replicationbut carry sequences, which allow their integration tochromosomes at high frequency. These plasmids arelinearized by a single restriction cut within thecomplementary yeast gene on the vector forintegrative gene conversion. A number of integratingvectors have been used successfully to express avariety of heterologous proteins in yeast. YIps withtwo different yeast DNA sequences, one coding forfunctional URA3 locus and the other coding for amutated "his3" gene, which is to be integrated into itsnormal 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 intochromosomal DNA (Scherer & Davis, 1979).

The second approach for the gene integration isgene replacement by homologous recombination. It isthe ability of complementary sequences, which alignand exchange the desired fragments in a doublecrossover event. There is exact base-to-base exchangein this process with no stop in the joints. Thefrequency of homologous recombination is muchgreater in yeast than in higher eukaryotes. Therefore,it has been exploited as one of the most importanttools in the yeast genetics. This approach has alsobeen used for the elimination of yeast genes that couldinterfere with efficient expression of desired foreignprotein. The transformation efficiency is very low(one transformant/ug DNAII 07_108 cells).

Replicating vectors. Yeast replicating vectors(YRps) contain prokaryotic plasmid DNA sequenceswith part of a yeast DNA derived from YIp vectorsand also include chromosomal origin of replication.The transformation efficiency of these plasmids is2-3 fold higher than the YIp vectors. This highfrequency is thought to be due to presence of origin ofreplication, which allows these vectors to replicateautonomously. 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 inlength (Stinchomb et al, 1979; Hitzeman et al, 1981).However, often a rapid loss of these plasmids is seenin growing cell population. Providing centromeric(CEN) DNA sequences to YRp plasmids can solvethis problem. These sequences contain a central ATrich region of 90 bp flanked by highly conservedregion 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 yeastmarker gene and the entire 2 ~ plasmid also known as"scp" plasmid. The 2 ~ plasmid is found in almost allstrains of S. cerevisiae in 50-100 copies per haploidcell amounting to 2-3% of total chromosomal DNA(Grimm et al, 1988; Smerdon et al, 1998). The moststriking structural feature of 2 ~ plasmid is thepresence of two inverted repeats of 599 bp whichdivide the plasmid molecule into two non-identicalregion. A recombination at these repeats yields twoforms (A and B) of this plasmid (Fig. 2). The 2 ~plasmid possesses a single origin of replication andreplicates autonomously (Harteley & Donelson, 1980;Smerdon et al, 1998).

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8gl11 Hind III

Eco RI (4361)

Barn HI (375)

Sal (651)

1 Eco RI2 Poll, dATP, dTIP/I3 Ligase (J

8gl11 Hind III

1 Hind III/Barn HI\2 Isolation of largefragment

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(2964) Form A

Eco RI A = 6318 - 2407 = 3912 bpEco RI B = 2407 bp

EcoRI(2407)

Hpa I(2964)

Form B

Eco RI A = 2407 - 341 + 2006 = 4072 bpEco RI B = 4312 - 2407 + 341 = 2246 bp

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


Artificial chromosome. Normal eukaryoticchromosomes are always linear. The plasmids, whensupplied with ARS, the CEN sequences and afunctional telomere, can get replicated in autonomousmanner and are stably maintained in linear form as anextra artificial mini-chromosome (Murray & Szostak,1986). While highly stable, these vectors are notuseful for expression of foreign proteins in yeast.

Expression System Using Yeast as a HostS. cerevisiae, the first species of yeast to be

employed for the production of recombinant proteins,has been used as conventional host for proteinproduction for research, industrial and medical uses(Romanos, 1995). Pichia pastoris and Hanselapolymorpha, both methylotrophic yeasts, wereoriginally developed for the large-scale and high-yieldproduction of heterologous proteins in a mediumcontaining methanol (Cregg et al, 1993; Bretthauer &Castellino 1999). Other commercial yeast strains,Kluyveromyces lactis, Yarrowia lipolytica andCandida utilitis (Fleer et al, 1991; Kondo et al, 1995),are phylogenetically closer to S. cerevisiae thanSchizosaccharomyces pombe. S. pombe is a promisinghost for an expression system as it often providesforeign gene products that are closer to their naturalform (Kaufer et al, 1985). Wild type yeasts areprototrophic i.e. they are nutritionally self-sufficientand capable of growing on minimal media.Auxotrophic yeast strains, created using classicalgenetics, provide the basis for selection ofsuccessfully transformed strains. By including a genein the plasmid expression cassette, that complementsone or more defective genes in the host auxotroph,one can easily select the recombinants on minimalmedia. Hence, strains requiring leucine (Leu-2) willgrow on minimal media if they harbour a plasmidexpressing the Leu-2 gene. The number and variety ofS. cerevisiae and S. pombe strains possessingnutritional markers make them attractive host, whilethey have some limitations also.

Methylotrophic YeastThe methylotrophic yeasts, H. polymorpha and P.

pastoris, can grow by utilizing methanol as the solesource of carbon and are fast becoming the favouriteyeast hosts for expression of cloned genes. P. pastorishas received widespread attention as an expressionsystem for its ability to express high level ofheterologous proteins (Sreekrishna et al, 1997;Higgins & Cregg, 1998; Cregg, 1999). Pichia caneasily be cultured, genetically manipulated and has a

secretary pathway very similar to mammalian cells.Both N-linked and a-linked glycosylation can takeplace. The complex glycoproteins, abundant with N-linked sites, expressed in Pichia are usuallybiologically active. It is possible to engineer theglycosylation pathway in Pichia to obtainglycoproteins very similar to those expressed inmammalian cells (Bretthauer & Castellino, 1999). Ofmore than120 heterologous proteins expressed in P.pastoris, most are of human and other mammalianorigin (Cregg, 1999). P. pastoris has two advantagesover S. cerevisiae. Firstly, its methanol-induciblealcohol oxidasel gene (AOXl) is tightly regulated,repressed in absence of methanol and induced ifmethanol is present (Tschopp et al, 1987). This canconveniently be used to drive production of toxicheterologous proteins, as it will not have any toxiceffect of heterologous protein to host until theexpression of the gene is induced by methanol(Cereghino & Cregg, 2000). The second advantage isthat 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 particularlyadvantageous for the production of therapeuticallyrelevant macromolecules in large amounts (Table 1)(Pichuantes et al, 1996; Hollenberg & Gellisen, 1997;Higgins & Cregg, 1998; Fischer et al, 1999;Cereghino & Cregg, 2000).

Prototrophic YeastS. pombe, a unicellular eukaryote belonging to the

ascomycests family, is called fission yeast as itreproduces by fission, besides through spores. UnlikeS. cerevisiae, no budding is observed in it. S. pombe isthe most intensively studied and well characterized

Table I-Expression of heterologous proteines in Pichia pastoris

Protein Reference( s)

Weiss et al, 1998Sahasrabudhe et al, 1998Sun et al, 1997Bromme et al, 1999Munshi et al, 1997Sen Gupta and Dighe, 1999

I3r Adrenergic receptorBile salt-stimulated lipaseCaspase-3Cathepsin VCD38Chorionic gonadotropin a-subunit,l3-subunit and al3-heterodimerInsulinInsulin like growth factor-I (IGF-l)Leukemia inhibitory factor (LIF)Lymphocyte surface antigen CD38Lysosomal u-manncsidaseMast cell tryptaseSerum albuminTumour necrosis factor (TNF) aCereghino & Cregg, 2000

Kjeldsen et al, 1999Brierley, 1998Zhang et al, 1997Fryxell et al, 1995Liao et al, 1996Chan et al, 1999Ohtani et al, 1998 a.bSreekrishna et al, 1989


yeast species than S. cerevisiae in terms of moleculargenetics and cell biology (Beach & Nurse, 1981;Russel, 1989). However, unlike other yeasts, S.pombe has many characteristics similar to highereukaryotic cells and it has not been used muchindustrially to make wine, beer and bread. It isgradually being considered as very usefulexperimental model for the study of molecularbiology of yeast. Some mammalian genes can beisolated using S. pombe by complementation of themutant homologue. The functional substitution ofhuman homologue of cell cycle regulator cdc2 for theS. pombe cdc2 gene (which is homologous to the S.cerevisiae CDC28 gene) is possible (Lee & Nurse,1987). The similarity of human cdc2 system and thatof S. pombe has been confirmed at the protein level.Some mammalian promoters are functional in S.pombe (Toyama & Okayama, 1990). Highereukaryotic genes containing introns when introducedinto S. cerevisiae are not expressed, whereas the samegenes can be expressed in S. pombe (Kaufer et al,1985). RNA splicing mechanism of S. pombe hasmore similarity with higher eukaryotes than with S.cerevisiae (Porter et al, 1990). A signal transductionsystem of S. pombe shows marked similarities tomammalian G-protein-coupled system (Xu et al,1994). The carbohydrate chains of yeast glycoproteinsare composed of N-linked and O-linked oligosac-charides with similar structure as in mammalian cells.However, generally the glycans of yeast have outerchains consisting of mainly mannose oligomers in N-linked and O-linked glycosylation in contrast to theglycoproteins derived from mammalian cells. S.pombe, unlike other yeast species, has galactoseresidues in mannose-rich sugar chains (Moreno et al,1990; Ballou et al, 1994). It can be considered uniqueyeast with characteristics closer to those ofmammalian cells making it an accurate model formolecular biology studies.

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Yeast Expression VectorsFor the construction of an expression system, after

deciding the host, an effectively functioningexpression vector containing necessary elements forthe selected host is constructed to suit high-levelexpression of the foreign gene. S. pombe moleculargenetics has led to greater understanding of thecomponents of each expression vector (Tohda et al,1994).

Chromosomal VectorsIn these vectors, a foreign gene is stably maintained

within a chromosome in the host genome. Someintegrating vectors currently available for use in S.pombe are based on complementation of S. pombemutations with S. cerevisiae genes including the leuland ura4 (Grimm et al, 1988) or on the integration of5 S ribosomal RNA gene (Smerdon et ai, 1998). Theanalysis of genomic DNA has shown that thetransformants have one or more copies of the gene ofinterest integrated into the host genome byhomologous recombination (Grallet et al, 1993).

Episomal VectorsThese vectors have the yeast origin of replication

and replicate extra-chromosomally in an autonomousmanner (Maundrell et al, 1988). In S. pombeexpression system, episomal vectors are commonlyused (Table 2). Following are the importantcomponents of these vectors:

i) Origin of replication. It is essential for DNAreplication. For autonomous episomal vectorsreplicating outside the chromosomes, the 2 !l ori orars l are commonly used (Losson & Lacroute, 1983).The 2 !l ori is the origin of replication of the 2 !lplasmid of the S. cerevisiae. The arsl, a region ofchromosomal DNA of S. pombe, was identified as oneof the autonomously replicating sequences (ARSs)and it promotes the extra-chromosomal autonomous

Vector Promoter Yeast markerOrigin of replication

Table 2-Expression vector systems in Shizosaccharomyces pombe

pART ars I adh promoter LEU 2pEVP II 2J..lplasmid ori adh promoter LEU 2pCHY21 ars I fbp I promoter URA 3pREP ars I nmt I promoter LEU 2pTL2M UpAL 7 ars II stb hCMV promoter neol LEU 21 URA 3pSL2M UpAL 7 ars II stb hCMV promoter neo/ LEU 21 URA 3pSLF ars I CaMV promoter LEU 2pSLF 101 ars I CaMV-tet LEU 2pSM 112 2J..lplasmid ori SV 40 promoter LEU 2pART IIN795 ars I adh promoter LEU 2

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


replication and maintenance of the plasmid outsidechromosomes (Wright et al, 1986). Plasmids havingS. pombe arsl are present in multiple copies (15-80),however, these are mitotically unstable. Inclusion ofstb sequences (stabilizing sequence) derived from S.pombe yield stable transformants both mitotically andmeiotically. When stb is combined with arsl, theresulting vector can be maintained more stably in S.pombe as a multiple copy vectors (up to 80copies/cell). The loss frequency of this plasmid isonly about 13% per generation without the selectionpressure (Wright et al, 1986). The ARS elements intransformants have been identified and have beenshown to co-localize with the chromosomal origin(CaddIe & Calos, 1994).

ii} Selection markers. The most extensively usedprotocols for transformation of S. pombe areelectoporation (Hood & Stachow, 1990), protoplastfusion (Beach & Nurse, 1981) and lithium acetatemediated uptake of plasmid DNA (Okazaki et al,1990). The lithium acetate method gives higherfrequency of transformation than the protoplast fusionprocedure. After transformation, the most commonlyused 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 selectablemarkers (Burke, 1994; Waddell & Jerkins, 1995;Cottarel, 1995). Although the sensitivity of S. pombeto various antibiotics is low, the arninoglycosideantibiotics G418, hygromycin, chloramphenicol,pleomycin and bleomycin can conveniently be used asselection markers (Burland et al. 1991). For selectablemarkers, genes have been described for moleculargenetic manipulation of P. pastoris. Existing markersare limited to the biosynthesis genes HIS 4 either fromP. pastoris or S. cerevisiae and ARG4 from S.cerevisiae (Cregg & Madden, 1989; Cereghino &Cregg, 2000).

iii) Promoter. Selection of a strong promoter iscritical for efficient expression. While manypromoters, including the promoters for the enzymesof glycolytic pathway are used in S. cerevisiaesystems, only limited numbers of promoters areknown to work efficiently in S. pombe. Successfulexpression of foreign genes in S. pombe has beenreported under the control of promoters for S.cerevisiae such as PGK55, ADHI and CYC (Belshamet al, 1986; Broker et al, 1987). A general expressionvector, pMB332 has been constructed for S. pombewhere the heterologous gene expression is driven by

adh promoter. The vector carries the URA3 gene of S.cerevisiae, which complements the S. pombe ura4mutation. The functional importance of this vectorsystem has been shown by the production of humanblood coagulation protein factor XIIIa (Broker &Baumal 1989). One of the efficient promoters ofglycolytic enzymes in S. pombe is the promoter ofadhI gene, coding for alcohol dehydrogenase.Vectors, pART and pEVPll, containing this promoterare widely used (Broker et al, 1987). Promotersderived from mammalian cells work efficiently withS. pombe. They include SV40 promoter (Jones et ai,1988), human cytomegalovirus (hCMV) promoter,human chorionic gonadotropin a-subunit promoter(Toyama & Okayama, 1990), adenovirus region 3promoter (Swaminathan et al, 1993), HIV-l longterminal repeat promoter (Toyama et al, 1992), andhuman 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 tofunction in S. pombe (Pobjecky et al, 1990; Truong etal, 1992). A number of inducible promoters in S.pombe, can be used effectively for the production oftoxic proteins. The most widely used induciblepromoter is nmtl promoter and an inducible vector,pREP, has been developed utilizing this promoter(Maundrell, 1990). The activity of this promoter issuppressed by thiamin. A system, regulated bytetracycline, combines the CaMV 35S promoter andtetracycline regulatory region of plasmid (Faryar &Gatz, 1992). Inducible promoters regulated byglucose concentration include the fbp l promoter(Hoffman & Winsten, 1989) and another promoter forinvl (Tanaka et al, 1998) coding for invertase in S.pombe. Foreign genes, including GFP (greenfluorescent protein), have been expressed successfullyutilizing these promoters (Tanaka et al, 1998). Giga-Hama (1997) constructed a high-level expressionvector (Fig. 3a), which is used along with atransducing vector (Fig. 3b) containing arsl/stb andLEU21URA3 genes. It is believed that following co-transformation homologous recombination betweenthese vectors takes place inside the cell and the twovectors replicate as a single plasmid.

Factors Involved in Homologous ExpressionYeasts are able to utilize many of the well-known

techniques used for the molecular and geneticmanipulation of growth characteristics in prokaryotes.Yeast also has the capability of complex post-translational modification. For the generation of


A SV40-T

stb

B

LEU2NRA3

Fig. 3-Structure of the expression vectors. A. High-levelexpression 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, originof replication; LEU21URA3, selection marker (Giga-Hama, 1997).

authentic recombinant proteins, following are theimportant events for the stability of heterologousexpression:

Post-translational ModificationsThe post-translational targeting and membrane

translocation pathways have also been demonstratedin S. cerevisiae (Rapoport et al, 1996a & b). Thecytoplasmic chaperone hsp 70 and its co-chaperonesmaintain the translocation competency of proteinsubstrates, although they do not possess targetingfunction. Genetically identified components have alsobeen functionally assessed using in vitroreconstitution systems. The studies demonstrated thata heptameric membrane complex (including twosubcomplexes) is essential for post-translationalprocesses (Panzner et al, 1995). One subcomplexconsists of four proteins (See 62p, See 63p, See 72p,and See 73p), while other subcomplex, See 61p, is atrimeric complex of See 61p, Sbh IP and Ssslp.

In contrast to the modification of the DNAsequence, each alteration introduced at thetranslational or post-translational level will be limitedto only one molecule and will not be furtheramplified. This can take place in a heterogeneousmixture of polypeptides. However, proteins withdiffering properties can only be readily detected, ifpresent in large amounts. Codon usage can playa keyrole in regulating gene expression and in theproduction of large quantities of high-qualityheterologous protein. At the translational level, errorscan occur due to missense insertions by tRNAs whoseanticodons match two out of three codon bases.Mistranslation can also occur if the mRNA from ahighly expressed heterologous gene contains rarecodons, or if the amino acid distribution were.inordinately skewed relative to typical yeast protein". ':

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The latter two possibilities can lead to ribosomalpausing and frame shifting, thus reducing quantity orquality of the desired gene product. Thus,optimization of the codons in a cloned gene to fit thepreferences of the yeast cell can reducemistranslation, although this is relatively simple forsmall proteins, it becomes more complex for largerproteins.

Sometimes authenticity of a mature protein mayrepresent a more important consideration than thehighest level of expression specially if the biologicalactivity and physiochemical properties such assolubility, biodistribution, circulatory half-life orstability are dependent on specific post-translationalmodifications. This is the case when heterologousgenes are expressed in cells that lack the appropriatemodification system, or if the production of aheterologous protein exceeds the post-translationalcapacity of the host cell. Many post-translationalmodifications of proteins are correctly done in yeastcells.

Intracellular ExpressionRecombinant proteins have been produced in yeast

both in intracellular and secretion expression systems.Intracellular expression is liked for heterologousproteins, which are normally expressed in thecytoplasm, as well as for those secreted proteins thathave no or relatively few disulphide bonds. Yeast cancarry out the post-translational removal of the initiatormethionine by the yeast methiony I aminopeptidasefrom the cytoplasmically expressed proteins. Wherethe amino-terminal methionine residue is retained,problems of immunogenicity may arise duringmedical applications. In addition to the removal of theamino-terminal methionine, yeast also carries outamino-terminal acetylation, carboxy-terminalmethylation, myristylation and farnesylation. Thelatter three are important in the membrane targeting ofintracellularly expressed proteins. Yeasts are alsocapable of assembling intracellularly expressedoligomeric proteins such as hepatitis B surfaceantigen (Valenzuala et al, 1982; Cregg et at, 1987;Janowicz et al, 1991) and the subunits of mammalianNa+, K+-ATPase (Eakle et al, 1992; Horowitz et al,1990). Hoffman et al (1.995) reported the high-levelexpression and functional assembly of the threehuman 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 thegeneral form, a2b2. The different chains werecorrectly processed at their amino termini and four


heme groups bound to the protein imitated the nativeprotein. An acetyl group blocks the amino-terminalamino acid of the recombinant chain, as is the case inthe naturally occurring molecule. The expression andprocessing of the hepatitis C virus core protein(HCcAg) were analyzed in methalotropic yeast, P.pastoris. The proteolytic processing of the precursorin P. pastoris produced two proteins (21 kDa and 23kDa), detected as the major product of HccAg, whichhave same N-terminal end and both react with amonoclonal antibody against the first 35 amino acidsof HCcAg. The correct proteolytic processing of therecombinant protein proves the usefulness of P.pastoris as an expression system to understand theprocessing of HCV structural proteins (Acosta-Riveroet al, 2002).

The ability of yeast to perform various post-translational modifications and targeting of proteins tospecific cell membranes has led to its evaluation as anexpression system for membrane proteins (Evans etal, 1995; Grisshammer & Tate, 1995). Membraneproteins require complex post-translationalmodifications including interaction with otherproteins, such as molecular chaperones, to attain theirfinal conformation and insertion into membranes. Dueto the poor understanding of the factors that areimportant for membrane protein insertion and folding,there are still only a few examples of highly expressedheterologous membrane proteins in yeast. Often, theinitial expression experiments are performed in a hostthat is homologous to the source of the membraneprotein. Plant and fungal membrane proteins are morereadily expressed in S. cerevisiae than the mammalianmembrane proteins (Villalba et al, 1992; Reutz &Gros, 1994; Grisshammer & Tate, 1995). However,there are some reports on expression of humanmembrane proteins, including the erythroid anionexchanger AEI (Sekler et al. 1995), the emopamil-binding protein (Hanner et al, 1995), the multipledrug 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) andestrogen (Arnold et al, 1996), and the Neurosporacrassa plasma membrane H+-ATPase (Mahanty &Searborrugh, 1996) by yeast. By using yeast system,sufficient amounts of functional protein suitable forbiochemical, pharmacological and biophysicalanalysis can be obtained. The vertebrateneuroendocrine peptide, cholecystokinin (CCK), issubjected to a number of post-translational

modifications for its maturation to biologically activeform. S. cervisiae was chosen as a model to study theevents related to endoproteolytic processing of pro-CCK. Expression of pro-CCK as a fusion protein tothe pre-pro leader peptide of a-mating factor directedthe protein through the secretary pathway and resultedin secretion of CCK with a glycine extension(Johnson et al, 2001). Higher levels of expression ofthe G-coupled human dopamine and mouse 5-HT5Aserotonin 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 offoreign proteins in yeast is generating substantialinterest in applications such as biocatalysis, whole-cell vaccines, and combinatorial library presentation(Schreuder et al, 1996).

The undetectable levels of direct cytoplasmicexpression of an authentic protein are relativelycommon problem. Yield and the biological activity ofa recombinant protein may be increased by fusing it toa rapidly folding protein such as thioredoxin, whichfacilitates its folding and enhances solubility. Analternate approach may be to fuse it to a peptide tag.which may be recognized by a commercially availablemonoclonal antibody (Kaslow et al, 1994). In suchinstances, the problem can be overcome by fusion ofthe desired protein to stable proteins such as humansuperoxide dismutase (hSOD) or human y-interferonCy-IFN). The hSOD fusion approach has been used tooverexpress a number of viral polypeptides fordiagnostic purposes (Kuo et al, 1989; Barr et ai,1987). These yeast-expressed proteins have beenpurified and incorporated into diagnostic kits for thescreening of blood for HIV -1 and hepatitis C virus(HCV) antibodies. Often, the fusion strategy results inan insoluble product that must be extracted andsubjected to an in vitro cleavage and refoldingprocess, which occurs with variable efficiency andmay be difficult for certain complex structuresresulting in low yield of a authentic product of high-quality. The difficulty of refolding and generation ofrecombinant proteins with authentic amino terminimay be overcome by their fusion to ubiquitin (Ub), ayeast hydrolase, a 76 amino acid protein derived fromthe processing of a larger precursors. Ub fusionexpression system takes advantage of the processingof the Ub precursor in vivo by an endogenous yeasthydrolase. The yeast Ub system, closely related to asimilar mammalian system, can be conveniently usedto express foreign proteins as fusion protein. When achimeric gene encoding an Ub:f3-galactosidase fusion


protein was expressed in yeast, highly stableexpression was obtained. Ub was later cleaved fromthe nascent fusion protein to obtain high yields of ~-galactosidase (Finley et at, 1984). When makingproducts intracellularly, the Ub fusion approach hastwo distinct advantages. First, it can significantlyenhance the yield and/or stability of proteins that areotherwise unstable in the cytosol, the amino-terminalUb moiety of the fusion possibly prevents immediatedegradation of the fusion partner (Ecker et at, 1989;Sabin et at, 1989). Secondly, apart from proline, it cangenerate a protein with any desired amino terminus,because Ub hydrolase processing is largelyindependent of the amino terminus of the proteinfused to Ub (Sabin et at, 1989).

Extracellular ExpressionThe secretion of heterologous proteins into the

culture medium offers a way to avoid toxicity fromaccumulated material and simplify purification of theprotein, because the yeast organism secretes relativelylow levels of native proteins. Furthermore, thepassage of the proteins through the secretory pathwaypermits post-translational events such as proteolyticmaturation, glycosylation and disulphide bondformation. The secretion of heterologous proteins isdriven by virtue of a cleavable, amino-terminal signalsequence that is derived from either the native proteinor the leaders of the S. cerevisiae prepro-mating factoror invertase. Numerous examples demonstrate thecapability of yeast to secrete mature humanpolypeptides possessing the expected biological, andin most cases, physico-chemical properties. Somesuch examples include the production of single chainFv 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) (Rosenfeldet at, 1996), analogs of tissue factor pathway inhibitor(Peterson et at, 1996), and interleukin-8 (Wernette-Hammond et at, 1996). In all these cases, specificstructure/function relationships such as catalyticactivity, ligand binding, ATP-dependent efflux,specific anti-idiotype binding and clot promotioncapacity were found to be comparable with the nativecounterpart. It is not surprising that yeast secretesnon-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 proteinthat could not be expressed successfully in bacterialor insect cell host systems, but was secreted in afunctional, authentic form by yeast.

Rational engineering can be used to produce novelproteins. P. pastoris has been used to efficientlyexpress rabbit Sc FV antibody fragment thatrecognizes human leukaemia inhibitory factor isolatedfrom a combinatorial library (Ridder et al, 1995).However, the removal of a signal sequence byspecific signal peptidase may sometimes be defectiveresulting in the production of a modified protein. It isreported that the removal of the a-factor leadersequences by the Kex2 protease (Kex2p) isincomplete, resulting in hyperglycosylated secretedmaterial with an amino-terminal extensions. Kjeldsenet at (1999) have shown that the introduction of anamino-terminal spacer between the a-factor leaderand the insulin precursor significantly improvedKex2p processing. The spacer peptide is thenremoved either in vitro by a specific protease or ill

vivo by the yeast aspartyl protease 3. Thismodification also increased fermenter yields of theinsulin precursor by 215%.

Protein Folding and SecretionThe secretion of properly folded proteins, crucial

for' correct biological activity, is one of the majorfactors for considering yeast as the preferred host forheterologous protein expression. This is because thedirect capture of active product from conditionedmedium eliminates the need for costly and low-yielding cell disruption or refold process steps.Although there are some exceptions, most notablyhuman serum albumin, which is secreted at 4 gl/ 1 byP. pastoris (Faber et at, 1995), the high productivityexpected from the high-copy number yeast vectorscontaining foreign genes driven by very strongpromoters is usually not obtained for secretedproteins. To better understand the mechanistic natureof this problem, Parekh et at (1995) have beenstudying bovine pancreatic trypsin inhibitor (BPTI) asa model of secretion and the role of folding in theendoplasmic reticulum (ER). In this system, the levelof expression driven by a multicopy (>50), 2/.l-basedplasmid vector was found equivalent to that driven bya single copy construct. There was higher genetranscription in the multicopy system. However, thebinding of conformationally specific antibodiesshowed that majority of the proteins in cellsharbouring the multicopy construct were improperly


folded and were retained in lumen of the ER(Robinson & Wittrup, 1995). This suggests thatmaintenance of correct intralumenal tertiary structuremay be one of the major factors for limitation ofexpression through the eukaryotic secretion pathway.

Many types of proteins could possibly be expressedin heterologous systems. Secretory proteins, onexpression, need to go through the reticulo-endothelial system for post-translational processing.The secretory signal, recognized by the signalpeptidase, is essential for passing through the correctsecretory pathway. Only a limited number of foreignsecretory proteins have been produced in S. pombe(Table 3). In case of human antithrombin II, humangastric lipase, human placental alkaline phosphataseand S. cerevisiae invertase, the secretory signals ofthe protein worked effectively in S. pombe, resultingin the secretion of the proteins (Broker et al, 1987).However, in case of many other secretory proteins,signal peptide is not recognized by the signalpeptidase of S. pombe, so the precursor proteins donot enter the endoplasmic reticulum but stay in thecell cytoplasm without undergoing maturation.

High expression of granulocyte-colony stimulatingfactor (G-CSF), erythropoietin (EPO) or S. pombeacid phosphatase in S. cerevisiae leads to low levelsof extractable heavy chain binding protein (BiP) andprotein disulphide isomerase (PDI), which are keyproteins involved in lumenal folding. By showing thatBiP synthesis rate was not reduced as a result of G-CSF expression and that augmented level of BiPcould not be restored by its co-expression, it wasgenerally suggested that foldase losses could be dueto degradation or aggregation (possibly as a result oftheir own misfolding) (Robinson & Wittrup, 1995)Unfortunately, the exact mechanism by which

Table 3-Production of heterologous proteins in S. pombe

Protein Reference

Human lipocortin IHuman blood coagulation factor XlllaB-GlucuronidaseHIV type-I protein RHuman Bradrenergic receptorHuman D2s dopamine receptorHuman P-glycoproteinHuman liver epoxide hydrolaseCytochrome P450Human gastric lipaseHuman antithrombin IIIHuman interleukin-6Human placental alkaline phosphataseGiga-Harna & Kumagai, 1999

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

lumenal proteins regulate the folding and secretion isnot fully understood and the productivity potential ofyeast remains underexploited.

GlycosylationIt is both, the most common and the most complex

form of post-translational modification (Meyniataalles& Combes, 1996). The majority of therapeuticallyrelevant proteins are usually glycoproteins in natureand must therefore be properly glycosylated to displaythe correct biological activity. Thus, the monitoring ofglycosylation patterns in quality control ofrecombinant therapeutic proteins to assure productsafety, efficacy and consistency is assurninz. b

importance. It is postulated that general function ofprotein glycosylation is to aid in the foldinz ofb

nascent polypeptide chain and in stabilization of theconformation of mature glycoprotein (Wang et al,

1996). The nature of polypeptide, the host-cellphenotype and the cell environment can determine theglycosylation pattern and hence the quality of theresulting glycoprotein.

Although a number of recombinant glycoproteinsof pharmaceutical or industrial value have beenobtained using yeast expression system, they oftenhave altered biological properties and functions ascompared to their native counterparts. This is mostlydue to differences in protein glycosylation. Alterationin normal glycosylation patterns of therapeuticproteins may affect their in vivo function in respect tosolubility, sensitivity to proteases, serum half-life, andbiological activities such as targeting to specific cellsor interaction with specific receptors. Yeast cellsrecognize the N-glycosylation recognition sequencesimilar to higher eukaryotic cells, indicating that theyhave the potential to glycosylate at the same sites asthe higher eukaryotic cells. However, thecarbohydrate moieties of yeast glycoproteinsprimarily consist of man nose residues appended indifferent linkages to the core glycosyl units. Therecombinant glycoproteins generated in S. cerevisiae,being high-rnannose type, will be recognized by themannose receptors on various cells and removed fromthe circulation when injected into mammalian species.In addition, non-human glycosylation patterns arepotentially immunoreactive to humans. Thehyperglycosylation very often dilutes the advantagesthat the microbial eukaryote S. cerevisiae might haveover E. coli or mammalian cell expression system.Mannan mutants, which exhibit less elaborate N-linked glycosylation, have been isolated. These


mutants as well as other yeast strains, however, do notgrow. Certain yeast species such as P. pastoris and H.polymorpha seem to be less prone tohyperglycosylating the heterologous proteins(Romanos et al, 1992; Stratton-Thomas et al, 1995).The average length of mannose residues in proteinsproduced in these two yeasts is only 8-14 monomers,compared to 50-100 monomers in proteins producedby S. cerevisiae (Giga-Hama & Hiromichi, 1999).Due to this problem, often the mammalian cells arethe preferred host cells for the generation ofrecombinant glycoproteins for therapeutics purposes.However, all the steps involved in eukaryotic proteintrafficking and post-translational modification maynot be same in yeast and the higher eukaryotes(Sadhukhan & Sen, 1996). This conclusion was drawnfrom the work done on testicular isozymes ofangiotensin-converting enzyme (ACET), where thecontributions of each of five potential N-glycosylationsites of ACET toward its synthesis, glycosylation,intracellular transport, cleavage secretion andenzymatic activity were studied. The a-linkedglycans of S. cerevisiae also differ from those ofhigher eukaryotes. Human urokinase plasminogenactivator (UPA) epidermal growth factor-like domainis post-translationally modified by an unusual 0-linked fucosy lation in both naturally isolated UPAand recombinant UPA produced in mammalianculture, but not in S. cerevisiae (Stratton-Thomaset al, 1995).

Until recently, some questions were raisedregarding the presence of a-linked glycans in Pichiaproteins. In general, such glycans are present but haverelatively shorter chain length than those found ineither S. cerevisiae or in mammalian system. Thesedifferences may not always influence biologicalactivity of the protein, but influence certain in vivoperformances such as the half-life of the protein.Though there are examples where differences inglycosylation of r-proteins did not result in anychange in in vivo performances. It may be possible toengineer the glycosylation pathway in P. pastoris toobtain glyco-conjugates having similarity with thestructure of proteins expressed in mammalian cells.There has been discussions about feasibility of havingsuch approaches for both mammalian and insect cells(Hogland et al, 1998; Hironaka et al, 1992; Kumar etal, 1996; Fusseneggar et al, 1999; Jarvis et al, 1999).

The ability to genetically engineer the yeast cellsand the increasing available knowledge of the hostmachinery necessary to form the complex

487

carbohydrate structures found on many mammalianproteins provides the opportunity to specificallyoptimize the host-cell background that will produceproteins of pharmaceutical interest with the desiredcarbohydrate structures. S. pombe gm 1+ gene encodesan UDP-galactose transporter for proteingalactosylation (Tabuchi et al, 1997; Tanaka et 01,2001). Further, mutant deficient in UDP-galactosetransport activity has been isolated (Takegawa et 01,1996; Tanaka et al, 2001). However, because it is notpossible to make meaningful generalizations about theoptimal glycosylation pattern of recombinanttherapeutics, each glycoprotein must be individuallyassessed in the context of both the systems in which itis expressed and the desired clinical benefit.

A certain degree of heterogeneity within arecombinant glycoprotein is permitted by regulatoryagencies provided that efficacy, safety andconsistency can be demonstrated. Glycosylationconsistency can also be monitored and comparativestudies of structure and function are becoming easierwith the development of automated and sensitive newtechniques (O'Neill, 1994; Garcia et al, 1995; Cregg,1999).

Improving Expression of Foreign ProteinsYeast can be grown as haploid, diploid or polyploid

cells. Cell size increases with increasing ploidy of thechromosomes. Haploid cells are favoured in basicresearch for genetic manipulation. However, use ofpolyploid yeast is less likely to result inspontaneously arising deleterious mutant phenotypesover many generations of growth. Strain stability is animportant' consideration in fermentation technology,since large-scale production is very sensitive toalterations in genetic background of the host strain.

Stable auxotropic mutants are essential for theselection of transformants in all yeast species (Cregget al, 1985; Dohmen et al, 1989). Deletions in the locifor the auxotropic markers are preferred to pointmutations. The genetic background of the host yeaststrain can affect the production and/or recovery of theheterologous proteins. The use of protease deficientstrains (Brierley et al, 1998; White et al, 1995) hasbeen crucial for the expression of a number ofauthentic mammalian (Barr et al, 1987) and viral(Bathurst et al, 1989) proteins. An additional proteasedeficient strain, SMD 1168~ pep4:: URA3~ Kex 1::SUC2 his4 ura3, inhibits proteolysis of murine andhuman endostatin (Boehm et al, 1999). Mutants, thatconfer resistance to the cloned genes, can be selected


for cytotoxicity prevented efficient protein expressionsuch as with IGF-l. Isolation of a class of yeast supersecreting mutants that support high level secretion offoreign proteins, have been achieved by mutagenesisof yeast strains followed by the screening of mutantsfor elevated protein secretion.

Generating Muiticopy StrainsOptimization of protein expression often includes

the isolation of multi copy expression strains, whichcontain multiple integrated copies of an expressioncassette and sometimes yield more heterologousprotein than a single copy strain (Clare et al, 1991).Three approaches led to multi copy expression strainsof P. pastoris. First approach involved theconstruction of a vector with multiple head-to-tailcopies of an expression cassette (Brierley, 1998). Aparticular advantage of this approach, especially inthe production of human pharmaceuticals, is that theprecise number of expression cassettes is known andcan be recovered for direct verification by DNAsequencing. Second method. utilizes expressionvectors containing the P. pastoris HIS 4 and bacterialTn903 Kan' genes. The bacterial kanamycinresistance gene also confers resistance to the relatedeukaryotic antibiotic G418 (Scorer et al, 1994). Thismethod results in subset of colonies enriched for thosecontaining multiple vector copies. However, thevector copy number varies greatly. Thus, a significantnumber (50-100) of transformants must be subjectedto further analysis of copy number and expressionlevel. The third approach to construct the multicopystrains involves the use of a vector with the bacterialhie gene, which confers resistance to the antibioticzeocin (Higgins et al, 1998). Unlike G418 selection,stains transformed with expression cassette containingthe zeocin marker can be selected directly byresistance to the drug. Additionally, plating onincreased concentration of zeocin in selected platescan enrich populations of the transformants formulticopy expression cassette strains. Also, as the shhie gene can serve as selectable marker in bothbacteria and yeast, these expression vectors arecompact and convenient to use.

High Cell Density Growth in Fermenter CulturesYeasts have many advantages of both the

prokaryotic and the eukaryotic systems. They arefaster, easier and less expensive to grow, the processcan be scaled up and the level of protein expression isvery high (10-100 fold) as compared to that of E. coli

(Faber et al, 1995; Vozza et al, 1996). Further, theprotein can be expressed as secretary protein, whichhelps in downstream processing and purification ofthe gene product.

P. pastoris, a relatively poor fermenter, may be anadvantage during industrial use, as in high cell densitycultures of S. cerevisiae, the ethanol (the product of S.cerevisiae fermentation) rapidly builds up to toxiclevels and hinders further growth of cell and theforeign protein production. With its preference forrespiratory growth, P. pastoris can be cultured atextremely high densities (500 A6oo1ml) undercontrolled environments of fermenters with little riskof 'pickling itself (Stratton et al, 1998). High-densitygrowth is especially important for secretary proteins,as the concentration of product in the medium isdirectly proportional to the concentration of cells inthe culture. Another positive aspect of growing P.pastoris in fermenter cultures is that the level oftranscription initiated from the AOXI promoter can be3-5 times greater in cells fed methanol at growthlimiting rates as compared to cells grown in excess ofmethanol.

A continuous fermentation process has beendeveloped for P. pastoris expression system. P.pastoris glyceraldehyde-3-phosphate dehydrogenasepromoter has been used to produce large quantities ofrecombinant human chitinase for pre-clinical studiesas a potential anti-fungal drug (Goodrick et al, 200 I).Expression levels of about 200 to 400 mg/ml havebeen demonstrated in fed-batch fermentation usingstrains with either the traditional methanol-inducibleor the constitutive GAP promoter. Proteolyticdegradation- of the enzymes was typically seen in fedbatch fermentation. No proteolytic degradation of theenzyme was seen in the continuous fermentationmode.

A hall mark of the P. pastoris system is the easewith which expression strains scale up from shakeflask to high density fermenter cultures. Althoughsome foreign proteins have expressed well in shakeflask cultures, expression levels are typically lowcompared to fermenter cultures (Gleeson et al, 1998;Clare et al, 1998). The growth conditions for P.pastoris are ideal for large-scale production ofheterologous protein because the medium componentsare in-expensive and defined, consisting of purecarbon source (glycerol and methanol), biotin, salts,trace elements and water. This medium is free ofundefined ingredients that can be sources of pyrogensor toxins and is, therefore, convenient for the


production of human pharmaceuticals. Also, as P.pastoris is cultured in media with a relatively low pHand methanol, it is less likely to become contaminatedwith most other microorganisms.

ConclusionsMany protein types could possibly be expressed in

heterologous systems. Only a limited number offoreign secretory proteins have been produced in S.pombe. The use of S. pombe for foreign geneexpression has a big advantage, as it has a welldeveloped Golgi apparatus and the galactosyltransferase enzyme system, that are not found in manyother yeasts. Most of the post-translationalmodifications and correct maturation of expressedproteins are, therefore, possible in S. pombe. Thegenome sequence of S. pombe (Wood et al, 2002) willprovide further insights in the development of the newexpression strategies for the high level expression ofproteins. The development of new generation ofexpression vectors has opened up a way for mass-production of proteins of biomedical importance in aneconomic manner using S. pombe. Hopefully, theadditional expression vectors for S. pombe will bedeveloped. However, S. pombe has somedisadvantages such as the choice of the vectors is stilllimited and the issue of biological activity ofexpressed proteins is unresolved. It is expected thatthese problem will soon be resolved and S. pombewill become one of the host of choice for high-levelexpression of many proteins.

AcknowledgementThe authors thank Mr A Mahesh and Mr P Gautam

for manuscript preparation. Financial assistance fromICAR as an ad hoc research project to S K J isgratefully acknowledged. M W N and V P are SeniorResearch Fellows of CSIR and ICMR, respectively.

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