Gene expression and engineering in yeast and other fungi Patricia Tekamp-Olson and Pablo Valenzuela C h i r o n C o r p o r a t i o n , 4560 H o r t o n Street, Emeryville, CA 94608, USA Current Opinion in Biotechnology 1990, 1:28-35
Introduction
Among yeast and other fungi, S a c c h a r o m y c e s cerevisiae has been the system of choice for molecular engineering because of the extensive biochemical, genetic, and fermentation knowledge attached to this yeast species. This knowledge and the similarity of the basic cellular processes of S. cerevisiae to those of higher eukaryotes have resulted in a well developed molecular engineering technology with applications in heterologous protein expression, strain development and in model system studies on gene organization, expression and replication in higher eukaryotes. We will summarize here the recent developments in S. cerevisiae expression systems and go on to discuss the recent advances in the engineering of yeast artificial chromosomes (YAC) for studies of higher eukaryotic gene organization and function. Finally, we will discuss expression in other yeast and fungi.
Saccharomyces cerevisiae The use of S. cerevisiae as a host system for the expression of recombinant proteins is well developed. Both integrative a n d episomal vectors are available and promoters that allow high-level constitutive and regulated expression have been well defined. Transformation is readily etfected by a variety of methods. The reader is referred to a recently published review [1 .°] that provides a general overview of expression. The success of S. cerevisiae as a host system for heterologous protein expression in the biopharmaceutical field is shown by a partial listing of proteins derived from recombinant yeast that are approved for sale or are being clinically evaluated (Table 1). These proteins are derived from both intracellular and secretion expression systems. The development of high-yield expression systems has been critical for the production of su~cient quantities of these factors to allow for the preclinical and clinical testing requisite for drug development. Secretion systems ate particularly suitable for heterologous protein expression because of the potential for: (1) obtaining the correct conformation in naturally se
creted proteins; (2) facilitating purification; and (3) allowing continuous fermentation. Scheckman and collaborators were the first to report genetic and biochemical evidence indicating that protein transport and secretion in S. cerevisiae were similar to that seen in higher eukaryotic cells [2]. During secretion, S. cerevisiae, like higher eukaryotes, carries out various post-translational modifications including proteolytic processing of preand propeptides [3] and glycosylation [4]. However, although the core structure of N-linked glycans from yeast and higher eukaryotes are identical, the outer chains differ in both composition and size. The O-linked glycans of S. cerevisiae also differ from those of higher eukaryotes. Gellerfors et al. [5 °] reported that some of the human insulin-like growth factor (IGF)-I secreted by S. cerevisiae is O-glycosylated, a modification that is not found in natural IGF-I. Thus, the possibility that yeast may glycosylate heterologous proteins differently from the native protein must be borne in mind, particularly when glycosylation is important for bioactivity or where antigenicity is a concern. Secretion systems for heterologous protein expression in S. cerevisiae have used both the native secretion signal or, more usually, a S. cerevisiae signal to mediate secretion. In particular, the signal/leader sequence of the yeast mating pheromone ¢~-factor, first introduced by Brake et al., has been very successful at directing the secretion of a wide variety of heterologous proteins [6,7]. Proteins with therapeutic potential recently secreted by such means include echistatin from snake venom, leech hirudin and human platelet-derived growth factor. Echistatin is a 49-amino-acid peptide with 8 cysteines and can inhibit platelet aggregation. Reported yields range from 0.1 to 0.5 mg/liter and the purified recombinant protein was identical to native echistatin in its ability to inhibit platelet aggregation [8]. Leech hirudin, which blocks coagulation, has previously been secreted using the S. cerevisiae (x-factor leader [9]. Riehl-Bellon et al. [10 °] report on the purification and characterization of this recombinant hirudin and show that is of comparable specific activity to natural hirudin, but lacks the sulfated tyrosine that is characteristic of native hirudin. Another commonly used secretion signal is the yeast invertase signal peptide. It has recently been employed to
Abbreviations ARS--autonomous replicating sequence; HIV--human immunodeficiency virus; IGF~insulin-like growth factor; YAC--yeast artificial chromosomes.
28
(~ Current Biology Ltd ISSN 0958-1669
Gene expression and engineering in yeast and other fungi Tekamp-Olson and Valenzuela direct the secretion of the 411-amino-acid, .single-chain urinary plasminogen activator (pro-urokinase) the gene of which was engineered to remove the site for N-linked glycosylation [11 .-]. Yields of up to 15 mg/liter of bioactive protein were achieved. This study used a 'super' secretion (sscl) strain of S. cerevisiaewhich may have been important for secretion of over two-thirds of this 47 000 kD protein into the culture medium. S. cerevisiae secretion expression systems have also been useful in the secretion and assembly of more complex proteins. Three homodimeric forms of human platelet-derived growth factor have been expressed in yeast and found to be fully active in a variety of assays [12]. The Torpedo californica nicotinic acetylcholine receptor, which is a pentameric protein comprising four independent subunits, a213~,8, has been expressed in yeast [13"]. Jansen et al. show that S. cerevisiae that has been engineered to carry integrated copies of all four subunit cDNAs produces stable mRNAs that include all the information necessary for functional expression of the nicotinic acetylcholine receptor in X e n o p u s oocytes. Western blotting experiments demonstrated the presence of novel proteins in recombinant yeast that have all the molecular and antigenic properties of authentic T. californica a, 13, 7 and 8 subunits. Unfortunately, these authors did not report on whether this recombinant receptor complex was functional. Choosing the best signal/leader sequence for the efficient secretion of any given heterologous protein must be done empirically. Although, in general, it has been found that secretion of heterologous protein is improved by the use of a yeast secretion signal [14], this is not always the case. Recently reported studies on S. cerevisiae secretion of proteins, including Aspergillus niger glucose oxidase [15"'], human 0t-amylase [16"] and human serum albumin [ 1 7 " ] , have shown that a mammalian signal may result in secretion levels equivalent to or greater than that achieved with a S. cerevisiae secretion signal. Furthermore, the choice of secretion signal can effect the efficiency of protein secretion as well as the quality of that protein [15"'-17"']. Finally, structure-function studies on an engineered signal sequence, the design of which was based on features common to signal peptides, have been used to define a generalized sequence for the efficient secretion of human lysozyme from S. cerevisiae [18,19]. It would be interesting to establish the e~ciency of this signal at mediating secretion of other heterologous proteins in yeast. An alternate strategy for expression in S. cerevisiae is intracellular expression. This strategy can result in the accumulation of very high levels of heterologous proteins; examples are shown in Table 1. Intracellular expression is the system of choice for heterologous proteins that are normally expressed in the cytoplasm as well as those secreted proteins that have no or very few disulfide bonds. An interesting example of intracellular expression is that of human Factor XIIIa, which is involved in the formation of stable blood clots. This 731-amino-acid protein has 9 cysteines, none of which appears to be involved in disulfide bonds. Rinas etal. [20.] have purified and characterized yeast-derived recombinant Factor XIIIa and shown
that it is identical to placental Factor XIIIa by a number of physiochemical and functional criteria. Like higher eukaryotes, yeast carry out post-translational modifications including removal of the N-terminal methionine, N-terminal acetylation, carboxyl-terminal methylation, myristylation and famesylation. The last three modifications are probably important in the membrane targeting of intracellularly expressed proteins. Jacobs et a/. [21-] have shown that the human immunodeficiency virus (H1V)-I gag precursor protein (p55), which is known to be naturally N-terminally myristylated, when synthesized in yeast is also myristylated and targeted to the plasma membrane. Mutant gag proteins that can not be N-terminally myristylated do not localize to the plasma membrane. Bathurst et al. [22] and Vlasuk et al. [23] have made similar observations for yeast expressed HIV1 p55; the latter group has also found that the purified recombinant p55 gag polyprotein is processed in vitro by HIV protease, but not by AMV protease, to yield authentic mature HIV core proteins. Carboxymethylation and farnesylation are other posttranslational modifications that have been implicated in membrane localization and function. Haploid yeast of the MATa mating type synthesize a 12-amino-acid mating factor that is not secreted by the classically defined secretory pathway. Anderegg et al. [24] have shown that a-factor is post-translationally modified by attachment of a famesyl residue to a terminal carboxymethylated cysteine. This is of interest because recent genetic and biochemical evidence indicates that post-translational processing of afactor and RAS proteins (guanine-binding proteins that participate in the control of eukaryotic cell proliferation) is the same and involves: (1) proteolytic removal of the three-carboxyl amino acids to generate a carboxyl-terminal cysteine; (2) carboxymethylation of the newly generated carboxyl-terminal cysteine; and (3) farnesylation of that cysteine [25",26,27]. Yeasts are also capable of assembling intracellularly expressed oligomeric proteins. In one of the earliest examples of heterologous protein expression in yeast, hepatitis-B surface antigen that had been expressed in S. cerevisiae was shown to be correctly assembled into particles indistinguishable from those in the serum of infected individuals [28]. More recently, Horowitz et al. [29"] reported the functional assembly in S. cerevisiae of the a and [3 subunits of mammalian Na,K +-ATPase, an integral membrane protein. In some cases, direct expression of heterologous polypeptides in S. cerevisiae results in the accumulation of low or undetectable levels of the desired protein. In such instances, fusion to a stably expressed protein, such as human superoxide dismutase, has been used successfully [30,31]. Such a strategy often results in the accumulation of large amounts of fusion protein; however, it is often insoluble and therefore the desired active protein must be extracted by solubilization, in vitro cleavage and refolding. While such a strategy is very useful for certain applications, such as the production of antigens for diagnostic purposes, there is room for improvement.
29
30
Expressionsystems
Table 1. Products by recombinant Saccharomyces cerevisiae approved for sale or in clinical trial Product
Use
Expression system
Reference
Recombivax HB®Engerix®
Hepatitis B vaccine
Intracellular
[28]
Malaria vivax CS protein
Malaria vaccine
Intracellular
Barr et al., J Exp Med 1987, 165:1160-1171
HIV-1 env2-3 ~rotein
HIV-1 vaccine
Intracellular
[31]
Human EGF
Soft tissue repair
Secretion
[6]
Human insulin
Treatment of diabetes
Secretion
Thim et al., Proc Natl Acad Sci USA 1986, 83:6766-6770
Human proinsulin
Treatment of diabetes
Intracellular
[30]
Human IGF-I
Treatment of catabolic states, osteoporosis
Secretion
[6]
Human SOD
Prevention of ischemic damage during tissue reperfusion
Intracellular
Hallewell et al.,Bio/ Technology 1987, 5:363-366
Human PDGF
Soft tissue repair
Secretion
[12]
Human FGF
Soft tissue repair
Intracellular
Barr eta/., J Biol Chem 1988, 263:16471-16478
Human G M - C S F
Treatment of neutropenia from chemo- and radiation therapy
Secretion
Cantrell et al., Proc Natl Acad Sci USA 1985, 82:6250-6254
Human o~lantitrypsin
Pulmonary emphysema
Intracellular
Rosenberg et al., Nature 1984, 312:77-80
HCV ELISA
Screening of blood for HCV antibodies
Intracellular
Kuo et al., Science 1989, 244:362-364
HIV ELISA
Screening of blood for HIV-1 antibodies
Intracellular
Steimer et al., In Vaccines 1987 Modern Approaches to New Vaccines edited by Chanock et al. Cold Spring Harbor, 1987, pp 326-341
RIBA-HIV-1
Recombinant immunoblot assay for HIV-1
Intracellular
Calarco et al., In Medical Virology VII edited by de la Maza and Peterson. Elsevier Science, 1988, pp 293-314
CS, circumsporozoite; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; SOD, superoxide dismutase.
Gene expression and engineering in yeast and other fungi Tekamp-O[son and Valenzuela An important recent development has been the description of a new intracellular fusion expression system that uses a yeast in vivo processing system to yield a product with a desired N-terminal amino acid, yet also gives equivalent or increased accumulation of heterologous protein relative to that achieved by direct expression. This expression system is based on the finding by Bachmair et al. [32] that a ubiquitin- ]3-galactosidase fusion protein, when expressed in yeast, was processed to yield free ubiquitin and [3-galactosidase irrespective of the identify of the first amino acid encoded by ]3-galactosidase gene (with the exception of proline). A number of groups have built on this observation and made ubiquitin fusions to a variety of heterologous proteins including: the 0t-subunit of the mammalian stimulating G-protein of the adenyl cylase complex [33"]; soluble T-cell receptor protein [33"]; the protease domain of human urokinase [33 "']; 7 interferon [34"']; % -proteinase inhibitor [ 3 4 " ] ; t h e 1,25(OH)2D 3 vitamin D receptor [35"']; and the chicken oviduct progesterone receptor [36"]. In all the above described examples, fusion to ubiquitin resuited in the production of fully processed fusion protein at levels at least equivalent to that observed with direct expression (when compared). The N-terminal amino acid of the cleaved heterologous protein was shown to be that expected for precise cleavage at the ubiquitin fusionprotein junction [34 "']. Furthermore, the expressed proteins were functional in the case of the ~z-subunit from the mammalian stimulating G-protein of the adenyl cyclase complex [33 "'], 7-interferon and ~zl-proteinase in hibitor [34"'], and 1,25(OH)2D 3 vitamin D and chicken oviduct progesterone receptors [35"',36"]. Thus, this ubiquitin fusion expression system will be a very useful system for the intracellular expression in yeast of high levels of recombinant proteins with authentic N-termini.
Yeast artificial chromosomes The extensive molecular and technical knowledge associated with S. cerevisiae prompted the development, in 1987, of a powerful new molecular engineering technol ogy - - that of YAC. Originally introduced as a cloning system for large pieces of DNA [37], it has recently found applications in the physical isolation of genes, the analysis of large transcription units of some mammalian genes and in the physical mapping and ordering of DNA segments from large genomes. Yeast artificial chromosomes are basically linear cloning vectors that supply a cloning site within a gene whose interruption is phenotypically visible in an appropriate yeast strain. They include a yeast autonomous replicating sequence (ARS) a yeast centromere, one selectable marker on each side of the centromere and, at both ends, sequences capable of inducing telomere formation in vivo. Important improvements and applications of this technology have been described during the last year. Traver et aL [38"'] have developed a rapid method for screening a YAC library using a yeast colony hybridization
method that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate; this allows direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. The authors also describe modifications of the YAC vector that allow the recovery of both ends of a given insert in E. colt plasmids and greatly facilitate walking within the YAC library. The inclusion of a marker for selection in mammalian cells allows the artificial replicon to be shuttied from yeast to mammalian cells for functional studies. Little et al. [39"] have shown that the YAC technology can be successfully applied to the isolation and study of specialized libraries from a given chromosome or from a selected portion of a given genome. They have shown that YAC libraries with DNA inserts as large as 800 kb can be generated from DNA isolated from human-hamster somatic cell hybrids in which the only human DNA is derived from the chromosomal region of interest (the human Xq24-Xq28 region). YACs containing human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human specific; the human DNA is not scrambled with hamster DNA. This result indicates that YAC technology will greatly facilitate the molecular analysis of specific human chromosomal regions for which appropriate somatic cell hybrids are available. Recently, the effects of the position of the ARS within the YAC have been examined [40 o]. Using Tetrahymena sequence as substrates for telemere formation, Wellinger and Zakin have shown that YACs with ARS on only one of their arms exhibit no differences in meiotic segregation and are no less stable than the corresponding constructs with ARS on both arms. This indicates that a functional centromere does not appear to act as a barrier to the movement of the replication fork. Yeast artificial chromosomes specific for a given human chromosome can be constructed by a method that uses flow-sorted chromosomes rather than hybrid cell lines as the DNA source [41]. This procedure, which was applied to the construction of a human chromosome-21-specific YAC, allows the use of nanogram to microgram quantities of DNA and minimizes the shearing of the genetic material by performing all the steps into low-melting-point agarose. Recently, Pavan et al. [42 "] have used fragmentation vectors to introduce a yeast telomere and selectable mark ers into yeast chromosomes by homologous recombination. A powerful application of this method uses the human Alu I repetitive sequence to target recombination to multiple independent sites on the human-derived YAC. This technique will probably have wide application in the future analysis, modification and selection of sequences cloned as YACs. The usefulness of the YAC technology in the construction of specific libraries and in the preparation of physical genomic maps has recently been applied to bacterial [43], Caenorhabditis [44], Drosophila [45], and human [46] genomes.
31
32
Expressionsystems Other yeast and fungi In addition to S. cerevisiae, there are many other yeast and fungal species that are important in the commercial production of enzymes, antibiotics and speciality chemicals, and as crop pathogens. The ability to engineer these yeast and other fungi would be very useful for the development of improved strains, heterologous protein production, and in the attenuation of fungal pathogens. In general, the ability to engineer these organisms has lagged behind that of S. cerevisiae, but progress is being made. The key to the genetic engineering and the development of heterologous expression systems in these other species is the availability of DNA transformation systems. Transformation has generally been effected by making cells permeable to DNA through enzymatic cell wall removal or by the use of high concentration of alkali cations. Variations of these methods have been successfully applied to a variety of fungal species [47-,]. Employing these classic methods, transformation of the following has been recently described: the filamentous yeast Trichosporon cutaneun, which may have potential for the efficient conversion of cheap carbon sources into biomass [48]; the industrial yeast Torulaspora delbruecki{ used in breadmaking and for secondary fermentation in the wine industry [49]; and the basidiomycete Phanerochaete chrysosporium, a lignin-degrading species [50]. Recently, very high efficiency transformation using electropulsation to introduce plasmid DNA into intact cells has been demonstrated in S. cerevisiae, Yarrowia lipolytica and Kluyveromyces lactis [51 o.]. This method may have general application to other fungal species. For many fungi, the development of transformation systems will facilitate molecular analysis and engineering and thus lead to strain improvements. The remainder of this section will focus on those other yeast and fungal species where the techniques for molecular engineering, especially for heterologous protein expression, are comparatively well developed. K lactis, a budding yeast like S. cerevisiae, has been in commercial use for years as a producer of the enzyme lactase. It is considered a 'safe' organism by the Food and Drug Administration which makes it attractive for the production of heterologous protein for food and drug use. Transformation of K lactis has been described [52]. Plasmid expression systems, based on a circular plasmid analogous to the 2 I.tm plasmid of S. cerevisiae, have also been described [53]. Van den Berg and collaborators [54 °°] have recently described the use of an integrative expression system employing the K lactis lactase promoter to express and secrete prochymosin from K lactis. Efficient and high-level secretion of fully active prochymosin was achleved
Introduction
Among yeast and other fungi, S a c c h a r o m y c e s cerevisiae has been the system of choice for molecular engineering because of the extensive biochemical, genetic, and fermentation knowledge attached to this yeast species. This knowledge and the similarity of the basic cellular processes of S. cerevisiae to those of higher eukaryotes have resulted in a well developed molecular engineering technology with applications in heterologous protein expression, strain development and in model system studies on gene organization, expression and replication in higher eukaryotes. We will summarize here the recent developments in S. cerevisiae expression systems and go on to discuss the recent advances in the engineering of yeast artificial chromosomes (YAC) for studies of higher eukaryotic gene organization and function. Finally, we will discuss expression in other yeast and fungi.
Saccharomyces cerevisiae The use of S. cerevisiae as a host system for the expression of recombinant proteins is well developed. Both integrative a n d episomal vectors are available and promoters that allow high-level constitutive and regulated expression have been well defined. Transformation is readily etfected by a variety of methods. The reader is referred to a recently published review [1 .°] that provides a general overview of expression. The success of S. cerevisiae as a host system for heterologous protein expression in the biopharmaceutical field is shown by a partial listing of proteins derived from recombinant yeast that are approved for sale or are being clinically evaluated (Table 1). These proteins are derived from both intracellular and secretion expression systems. The development of high-yield expression systems has been critical for the production of su~cient quantities of these factors to allow for the preclinical and clinical testing requisite for drug development. Secretion systems ate particularly suitable for heterologous protein expression because of the potential for: (1) obtaining the correct conformation in naturally se
creted proteins; (2) facilitating purification; and (3) allowing continuous fermentation. Scheckman and collaborators were the first to report genetic and biochemical evidence indicating that protein transport and secretion in S. cerevisiae were similar to that seen in higher eukaryotic cells [2]. During secretion, S. cerevisiae, like higher eukaryotes, carries out various post-translational modifications including proteolytic processing of preand propeptides [3] and glycosylation [4]. However, although the core structure of N-linked glycans from yeast and higher eukaryotes are identical, the outer chains differ in both composition and size. The O-linked glycans of S. cerevisiae also differ from those of higher eukaryotes. Gellerfors et al. [5 °] reported that some of the human insulin-like growth factor (IGF)-I secreted by S. cerevisiae is O-glycosylated, a modification that is not found in natural IGF-I. Thus, the possibility that yeast may glycosylate heterologous proteins differently from the native protein must be borne in mind, particularly when glycosylation is important for bioactivity or where antigenicity is a concern. Secretion systems for heterologous protein expression in S. cerevisiae have used both the native secretion signal or, more usually, a S. cerevisiae signal to mediate secretion. In particular, the signal/leader sequence of the yeast mating pheromone ¢~-factor, first introduced by Brake et al., has been very successful at directing the secretion of a wide variety of heterologous proteins [6,7]. Proteins with therapeutic potential recently secreted by such means include echistatin from snake venom, leech hirudin and human platelet-derived growth factor. Echistatin is a 49-amino-acid peptide with 8 cysteines and can inhibit platelet aggregation. Reported yields range from 0.1 to 0.5 mg/liter and the purified recombinant protein was identical to native echistatin in its ability to inhibit platelet aggregation [8]. Leech hirudin, which blocks coagulation, has previously been secreted using the S. cerevisiae (x-factor leader [9]. Riehl-Bellon et al. [10 °] report on the purification and characterization of this recombinant hirudin and show that is of comparable specific activity to natural hirudin, but lacks the sulfated tyrosine that is characteristic of native hirudin. Another commonly used secretion signal is the yeast invertase signal peptide. It has recently been employed to
Abbreviations ARS--autonomous replicating sequence; HIV--human immunodeficiency virus; IGF~insulin-like growth factor; YAC--yeast artificial chromosomes.
28
(~ Current Biology Ltd ISSN 0958-1669
Gene expression and engineering in yeast and other fungi Tekamp-Olson and Valenzuela direct the secretion of the 411-amino-acid, .single-chain urinary plasminogen activator (pro-urokinase) the gene of which was engineered to remove the site for N-linked glycosylation [11 .-]. Yields of up to 15 mg/liter of bioactive protein were achieved. This study used a 'super' secretion (sscl) strain of S. cerevisiaewhich may have been important for secretion of over two-thirds of this 47 000 kD protein into the culture medium. S. cerevisiae secretion expression systems have also been useful in the secretion and assembly of more complex proteins. Three homodimeric forms of human platelet-derived growth factor have been expressed in yeast and found to be fully active in a variety of assays [12]. The Torpedo californica nicotinic acetylcholine receptor, which is a pentameric protein comprising four independent subunits, a213~,8, has been expressed in yeast [13"]. Jansen et al. show that S. cerevisiae that has been engineered to carry integrated copies of all four subunit cDNAs produces stable mRNAs that include all the information necessary for functional expression of the nicotinic acetylcholine receptor in X e n o p u s oocytes. Western blotting experiments demonstrated the presence of novel proteins in recombinant yeast that have all the molecular and antigenic properties of authentic T. californica a, 13, 7 and 8 subunits. Unfortunately, these authors did not report on whether this recombinant receptor complex was functional. Choosing the best signal/leader sequence for the efficient secretion of any given heterologous protein must be done empirically. Although, in general, it has been found that secretion of heterologous protein is improved by the use of a yeast secretion signal [14], this is not always the case. Recently reported studies on S. cerevisiae secretion of proteins, including Aspergillus niger glucose oxidase [15"'], human 0t-amylase [16"] and human serum albumin [ 1 7 " ] , have shown that a mammalian signal may result in secretion levels equivalent to or greater than that achieved with a S. cerevisiae secretion signal. Furthermore, the choice of secretion signal can effect the efficiency of protein secretion as well as the quality of that protein [15"'-17"']. Finally, structure-function studies on an engineered signal sequence, the design of which was based on features common to signal peptides, have been used to define a generalized sequence for the efficient secretion of human lysozyme from S. cerevisiae [18,19]. It would be interesting to establish the e~ciency of this signal at mediating secretion of other heterologous proteins in yeast. An alternate strategy for expression in S. cerevisiae is intracellular expression. This strategy can result in the accumulation of very high levels of heterologous proteins; examples are shown in Table 1. Intracellular expression is the system of choice for heterologous proteins that are normally expressed in the cytoplasm as well as those secreted proteins that have no or very few disulfide bonds. An interesting example of intracellular expression is that of human Factor XIIIa, which is involved in the formation of stable blood clots. This 731-amino-acid protein has 9 cysteines, none of which appears to be involved in disulfide bonds. Rinas etal. [20.] have purified and characterized yeast-derived recombinant Factor XIIIa and shown
that it is identical to placental Factor XIIIa by a number of physiochemical and functional criteria. Like higher eukaryotes, yeast carry out post-translational modifications including removal of the N-terminal methionine, N-terminal acetylation, carboxyl-terminal methylation, myristylation and famesylation. The last three modifications are probably important in the membrane targeting of intracellularly expressed proteins. Jacobs et a/. [21-] have shown that the human immunodeficiency virus (H1V)-I gag precursor protein (p55), which is known to be naturally N-terminally myristylated, when synthesized in yeast is also myristylated and targeted to the plasma membrane. Mutant gag proteins that can not be N-terminally myristylated do not localize to the plasma membrane. Bathurst et al. [22] and Vlasuk et al. [23] have made similar observations for yeast expressed HIV1 p55; the latter group has also found that the purified recombinant p55 gag polyprotein is processed in vitro by HIV protease, but not by AMV protease, to yield authentic mature HIV core proteins. Carboxymethylation and farnesylation are other posttranslational modifications that have been implicated in membrane localization and function. Haploid yeast of the MATa mating type synthesize a 12-amino-acid mating factor that is not secreted by the classically defined secretory pathway. Anderegg et al. [24] have shown that a-factor is post-translationally modified by attachment of a famesyl residue to a terminal carboxymethylated cysteine. This is of interest because recent genetic and biochemical evidence indicates that post-translational processing of afactor and RAS proteins (guanine-binding proteins that participate in the control of eukaryotic cell proliferation) is the same and involves: (1) proteolytic removal of the three-carboxyl amino acids to generate a carboxyl-terminal cysteine; (2) carboxymethylation of the newly generated carboxyl-terminal cysteine; and (3) farnesylation of that cysteine [25",26,27]. Yeasts are also capable of assembling intracellularly expressed oligomeric proteins. In one of the earliest examples of heterologous protein expression in yeast, hepatitis-B surface antigen that had been expressed in S. cerevisiae was shown to be correctly assembled into particles indistinguishable from those in the serum of infected individuals [28]. More recently, Horowitz et al. [29"] reported the functional assembly in S. cerevisiae of the a and [3 subunits of mammalian Na,K +-ATPase, an integral membrane protein. In some cases, direct expression of heterologous polypeptides in S. cerevisiae results in the accumulation of low or undetectable levels of the desired protein. In such instances, fusion to a stably expressed protein, such as human superoxide dismutase, has been used successfully [30,31]. Such a strategy often results in the accumulation of large amounts of fusion protein; however, it is often insoluble and therefore the desired active protein must be extracted by solubilization, in vitro cleavage and refolding. While such a strategy is very useful for certain applications, such as the production of antigens for diagnostic purposes, there is room for improvement.
29
30
Expressionsystems
Table 1. Products by recombinant Saccharomyces cerevisiae approved for sale or in clinical trial Product
Use
Expression system
Reference
Recombivax HB®Engerix®
Hepatitis B vaccine
Intracellular
[28]
Malaria vivax CS protein
Malaria vaccine
Intracellular
Barr et al., J Exp Med 1987, 165:1160-1171
HIV-1 env2-3 ~rotein
HIV-1 vaccine
Intracellular
[31]
Human EGF
Soft tissue repair
Secretion
[6]
Human insulin
Treatment of diabetes
Secretion
Thim et al., Proc Natl Acad Sci USA 1986, 83:6766-6770
Human proinsulin
Treatment of diabetes
Intracellular
[30]
Human IGF-I
Treatment of catabolic states, osteoporosis
Secretion
[6]
Human SOD
Prevention of ischemic damage during tissue reperfusion
Intracellular
Hallewell et al.,Bio/ Technology 1987, 5:363-366
Human PDGF
Soft tissue repair
Secretion
[12]
Human FGF
Soft tissue repair
Intracellular
Barr eta/., J Biol Chem 1988, 263:16471-16478
Human G M - C S F
Treatment of neutropenia from chemo- and radiation therapy
Secretion
Cantrell et al., Proc Natl Acad Sci USA 1985, 82:6250-6254
Human o~lantitrypsin
Pulmonary emphysema
Intracellular
Rosenberg et al., Nature 1984, 312:77-80
HCV ELISA
Screening of blood for HCV antibodies
Intracellular
Kuo et al., Science 1989, 244:362-364
HIV ELISA
Screening of blood for HIV-1 antibodies
Intracellular
Steimer et al., In Vaccines 1987 Modern Approaches to New Vaccines edited by Chanock et al. Cold Spring Harbor, 1987, pp 326-341
RIBA-HIV-1
Recombinant immunoblot assay for HIV-1
Intracellular
Calarco et al., In Medical Virology VII edited by de la Maza and Peterson. Elsevier Science, 1988, pp 293-314
CS, circumsporozoite; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; SOD, superoxide dismutase.
Gene expression and engineering in yeast and other fungi Tekamp-O[son and Valenzuela An important recent development has been the description of a new intracellular fusion expression system that uses a yeast in vivo processing system to yield a product with a desired N-terminal amino acid, yet also gives equivalent or increased accumulation of heterologous protein relative to that achieved by direct expression. This expression system is based on the finding by Bachmair et al. [32] that a ubiquitin- ]3-galactosidase fusion protein, when expressed in yeast, was processed to yield free ubiquitin and [3-galactosidase irrespective of the identify of the first amino acid encoded by ]3-galactosidase gene (with the exception of proline). A number of groups have built on this observation and made ubiquitin fusions to a variety of heterologous proteins including: the 0t-subunit of the mammalian stimulating G-protein of the adenyl cylase complex [33"]; soluble T-cell receptor protein [33"]; the protease domain of human urokinase [33 "']; 7 interferon [34"']; % -proteinase inhibitor [ 3 4 " ] ; t h e 1,25(OH)2D 3 vitamin D receptor [35"']; and the chicken oviduct progesterone receptor [36"]. In all the above described examples, fusion to ubiquitin resuited in the production of fully processed fusion protein at levels at least equivalent to that observed with direct expression (when compared). The N-terminal amino acid of the cleaved heterologous protein was shown to be that expected for precise cleavage at the ubiquitin fusionprotein junction [34 "']. Furthermore, the expressed proteins were functional in the case of the ~z-subunit from the mammalian stimulating G-protein of the adenyl cyclase complex [33 "'], 7-interferon and ~zl-proteinase in hibitor [34"'], and 1,25(OH)2D 3 vitamin D and chicken oviduct progesterone receptors [35"',36"]. Thus, this ubiquitin fusion expression system will be a very useful system for the intracellular expression in yeast of high levels of recombinant proteins with authentic N-termini.
Yeast artificial chromosomes The extensive molecular and technical knowledge associated with S. cerevisiae prompted the development, in 1987, of a powerful new molecular engineering technol ogy - - that of YAC. Originally introduced as a cloning system for large pieces of DNA [37], it has recently found applications in the physical isolation of genes, the analysis of large transcription units of some mammalian genes and in the physical mapping and ordering of DNA segments from large genomes. Yeast artificial chromosomes are basically linear cloning vectors that supply a cloning site within a gene whose interruption is phenotypically visible in an appropriate yeast strain. They include a yeast autonomous replicating sequence (ARS) a yeast centromere, one selectable marker on each side of the centromere and, at both ends, sequences capable of inducing telomere formation in vivo. Important improvements and applications of this technology have been described during the last year. Traver et aL [38"'] have developed a rapid method for screening a YAC library using a yeast colony hybridization
method that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate; this allows direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. The authors also describe modifications of the YAC vector that allow the recovery of both ends of a given insert in E. colt plasmids and greatly facilitate walking within the YAC library. The inclusion of a marker for selection in mammalian cells allows the artificial replicon to be shuttied from yeast to mammalian cells for functional studies. Little et al. [39"] have shown that the YAC technology can be successfully applied to the isolation and study of specialized libraries from a given chromosome or from a selected portion of a given genome. They have shown that YAC libraries with DNA inserts as large as 800 kb can be generated from DNA isolated from human-hamster somatic cell hybrids in which the only human DNA is derived from the chromosomal region of interest (the human Xq24-Xq28 region). YACs containing human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human specific; the human DNA is not scrambled with hamster DNA. This result indicates that YAC technology will greatly facilitate the molecular analysis of specific human chromosomal regions for which appropriate somatic cell hybrids are available. Recently, the effects of the position of the ARS within the YAC have been examined [40 o]. Using Tetrahymena sequence as substrates for telemere formation, Wellinger and Zakin have shown that YACs with ARS on only one of their arms exhibit no differences in meiotic segregation and are no less stable than the corresponding constructs with ARS on both arms. This indicates that a functional centromere does not appear to act as a barrier to the movement of the replication fork. Yeast artificial chromosomes specific for a given human chromosome can be constructed by a method that uses flow-sorted chromosomes rather than hybrid cell lines as the DNA source [41]. This procedure, which was applied to the construction of a human chromosome-21-specific YAC, allows the use of nanogram to microgram quantities of DNA and minimizes the shearing of the genetic material by performing all the steps into low-melting-point agarose. Recently, Pavan et al. [42 "] have used fragmentation vectors to introduce a yeast telomere and selectable mark ers into yeast chromosomes by homologous recombination. A powerful application of this method uses the human Alu I repetitive sequence to target recombination to multiple independent sites on the human-derived YAC. This technique will probably have wide application in the future analysis, modification and selection of sequences cloned as YACs. The usefulness of the YAC technology in the construction of specific libraries and in the preparation of physical genomic maps has recently been applied to bacterial [43], Caenorhabditis [44], Drosophila [45], and human [46] genomes.
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Expressionsystems Other yeast and fungi In addition to S. cerevisiae, there are many other yeast and fungal species that are important in the commercial production of enzymes, antibiotics and speciality chemicals, and as crop pathogens. The ability to engineer these yeast and other fungi would be very useful for the development of improved strains, heterologous protein production, and in the attenuation of fungal pathogens. In general, the ability to engineer these organisms has lagged behind that of S. cerevisiae, but progress is being made. The key to the genetic engineering and the development of heterologous expression systems in these other species is the availability of DNA transformation systems. Transformation has generally been effected by making cells permeable to DNA through enzymatic cell wall removal or by the use of high concentration of alkali cations. Variations of these methods have been successfully applied to a variety of fungal species [47-,]. Employing these classic methods, transformation of the following has been recently described: the filamentous yeast Trichosporon cutaneun, which may have potential for the efficient conversion of cheap carbon sources into biomass [48]; the industrial yeast Torulaspora delbruecki{ used in breadmaking and for secondary fermentation in the wine industry [49]; and the basidiomycete Phanerochaete chrysosporium, a lignin-degrading species [50]. Recently, very high efficiency transformation using electropulsation to introduce plasmid DNA into intact cells has been demonstrated in S. cerevisiae, Yarrowia lipolytica and Kluyveromyces lactis [51 o.]. This method may have general application to other fungal species. For many fungi, the development of transformation systems will facilitate molecular analysis and engineering and thus lead to strain improvements. The remainder of this section will focus on those other yeast and fungal species where the techniques for molecular engineering, especially for heterologous protein expression, are comparatively well developed. K lactis, a budding yeast like S. cerevisiae, has been in commercial use for years as a producer of the enzyme lactase. It is considered a 'safe' organism by the Food and Drug Administration which makes it attractive for the production of heterologous protein for food and drug use. Transformation of K lactis has been described [52]. Plasmid expression systems, based on a circular plasmid analogous to the 2 I.tm plasmid of S. cerevisiae, have also been described [53]. Van den Berg and collaborators [54 °°] have recently described the use of an integrative expression system employing the K lactis lactase promoter to express and secrete prochymosin from K lactis. Efficient and high-level secretion of fully active prochymosin was achleved
