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Evaluation of Yeast as an Expression System

Indian Journal of Biotechnology Vol 2, October 2003, pp 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
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Indian Journal of Biotechnology Vol 2, October 2003, pp 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 , 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: ; Fax: ). 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, OCTOBER 2003 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 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 479 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 0 7 _10 8 cells). 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 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). Eco RI (4361) 1 Eco RI 2 Poll, datp, dtip/i 3 Ligase (J 8gl11 Hind III 1 Hind III/Barn HI\ 2 Isolation of large fragment 8gl11 Hind III 8gl11 Hind III Barn HI (375) Sal (651) Barn HI (375) Sal (651) Fig. I-Construction of Yrp7 vector and its derivative pfrl4 (Hitzeman et al, 1981) Eco RI (2407) EcoRI (2407) Hpa I (2964) Form A Hpa I (2964) Eco RI A = = 3912 bp Eco RI B = 2407 bp Form B Eco RI A = = 4072 bp Eco RI B = = 2246 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, OCTOBER 2003 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 secretary pathway very similar to mammalian cells. Both N-linked and a-linked glycosylation can take place. The complex glycoproteins, abundant with N- linked 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 481 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 N- linked 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 vector systems in Shizosaccharomyces pombe 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
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