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Volume 6 Number 4, July/August 1996,pp.230-237 PRODUCTION OF THERAPEUTIC PROTEINS IN YEASTS: A REVIEW Prasad Rallabhandi and Pak-Lam Yu, Biotechnology Group, Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand About the authors: Mr P. Rallabhandi is a Ph.D. student and Dr P.-L. Yu is a Senior Lecturer at the Department of Process and Environmental Technology, Masssey University. Further enquiries to Dr P.-L. Yu
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[ALL TABLES AT END OF TEXT] ABSTRACT This paper reviews the use of yeasts in the production of heterologous proteins with a special focus on therapeutic proteins. Yeast offers a number of advantages as an expression system over prokaryotic systems. Yeasts are eukaryotic in nature with consequent associated cellular processes such as protein secretion and post-translational modification of proteins to render the recombinant proteins biologically active. This is essential for the naturally secreted therapeutic proteins. With the combination of recombinant techniques and fermentation technology, it is possible to explore yeasts as hosts for the expression and secretion of medically important proteins.
INTRODUCTION Recombinant DNA technology has enabled the expression of many heterologous genes to produce medically and/or industrially important proteins in various host systems, enabling the large scale production of proteins, which cannot be produced otherwise in significant amounts from natural sources. Early successes in recombinant protein production were achieved using Escherichia coli as a host. Although recombinant proteins expressed in E.coli can reach very high levels (commonly 10-20% of total cell protein), a number of limitations exist with regard to the usefulness of such a host system (Harris, 1983). One major drawback associated with this bacterium is that, in most cases, it is incapable of secreting the heterologous proteins into the culture medium. Consequently, recombinant proteins accumulate intracellularly as insoluble and inactive form of inclusion bodies. Sophisticated and expensive techniques are then needed to convert these inclusion bodies to soluble and active products, usually involving denaturation agents for solubilisation, followed by complicated controlled refolding (Martson, 1986). Since most pharmacologically important proteins are naturally secreted, they often can only adopt their correct conformation by folding within the secretory pathway (Romanos et al., 1992). So, if they are synthesized and accumulated in cytoplasm like in E.coli, they do not often fold correctly and hence are not bioactive. In general, secretion is required for therapeutic proteins to form disulfide bonds and hence correctly folded in order to be bioactive. Furthermore, E.coli produces endotoxins and pyrogens which can contaminate the desired recombinant protein produced. So, when the end product is used as a therapeutic product, great care must be taken in the purification steps to remove the residual endotoxins and DNA. Also E.coli lacks the necessary cellular machinery to perform post-translational protein modifications which are specific to eukaryotic organisms, including yeasts and humans (Carter et al., 1987). These drawbacks with E.coli system have been the major driving forces for the development of yeast as an alternative host for heterologous gene expression especially for therapeutic products.
Yeast Hosts Since the discovery that yeast cells could be transformed with exogenous DNA (Beggs, 1978; Hinnen, 1978; Struhl et al., 1979), there has been much interest in genetic manipulation of yeasts for the synthesis of recombinant proteins. The majority of heterologous proteins produced in yeast have been expressed using Saccharomyces cerevisiae as a host system (Table 1). This is because of the wealth of knowledge that had been accumulated about its genetics and physiology, being a widely used organism in baking, brewing, wine making and distillary industries (Russel et al., 1991). However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming popular as hosts for recombinant protein production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolitica. The performance of these alternative yeast expression systems was reviewed by Buckholz and Gleeson, 1991. Examples of therapeutic proteins expressed from various non-Saccharomyces yeasts are shown in Table 2.
Advantages of Yeast Host Systems A primary important attribute is that yeasts are eukaryotic organisms and have a similar cellular structure and biochemical composition to other eukaryotes. This provides for post-translational modification of protein products such as disulfide bond formation, endoproteolytic cleavage, glycosylation and multimeric assembly required to produce authentic and bioactive mammalian proteins. Yet, yeasts retain the advantages of unicellular microorganisms with respect to rapid growth and ease of genetic manipulation. Yeasts of Saccharomyces and Kluyveromyces genus are devoid of endotoxins and classified as GRAS (Generally Regarded As Safe) microorganisms which represents an important factor for the production of pharmaceutical grade products. Greater progress and ready public acceptability have been achieved in the area of pharmaceutical biotechnology. Many biomedical products such as peptide hormones, blood proteins and recombinant vaccines have been produced from yeasts and are in current therapeutic uses (Tables 1 & 2). Factors such as easy cultivation, highly controllable fermentation in defined conditions and containment make yeasts ideal hosts for pharmaceutical production. The first genetically engineered vaccine licensed by Food and Drug Administration (FDA), U.S.A., for administration to humans, Hepatitis B Surface Antigen (HBsAg) was produced in Saccharomyces cerevisiae (Valenzuela, 1982). Further optimization of the HBsAg production to industrial scale has been reported (Fu et al., 1996a; Clarke et al., 1987). A number of other therapeutic proteins (eg. hirudin, insulin-like growth factor) expressed in Saccharomyces cerevesiae are in pre-clinical or clinical trials. Another important factor with yeasts is that they are able to secrete certain protein products into the extracellular environment in basically the same way as higher animal and plant cells. Secretion of heterologous proteins is preferred than having them synthesized and accumulated in cytoplasm for the following reasons. 1) Some foreign proteins may be toxic to yeast. 2) Some proteins are themselves secreted and processed from a large precursor without the usual methionine at the NH2-terminal of the mature protein eg., mature hirudin (Loison et al., 1988). Many biotechnology products of biomedical or veterinary importance are naturally secreted peptides or proteins, for which production in yeast secretory pathway is ideally suited (Romanos et al., 1992). Since yeasts normally secrete only a few proteins at low abundance, so that recombinant protein can be produced away from the majority of yeast proteins, which are retained within the cell, to give an advantage for their recovery and purification (Hadfield et al., 1993). 3) Many proteins contain internal disulfide bonds. The cytoplasm of yeast is a reducing environment which prevents the accurate formation of these bonds. On the other hand, when a protein is secreted through the yeast secretory pathway, disulfide bonds appear to be correctly formed, which is similar to that in higher eukaryotes (Hadfield et al., 1993). Yeast secretional vectors are available with expression cassette (complex) which consists of a promoter, a secretion signal coding sequence, cloning site(s) and a terminator. The preproleader region of the yeast alpha-factor pheromone has been extensively used for secreting heterologous proteins (Brake et al., 1984). Other commonly used signal peptides are MF alpha-pre (Siddu & Bollon, 1987), acid phosphatase (Izumoto, 1987), invertase (Smith et al., 1985), Kluyveromyces lactis killer toxin (Livi et al., 1990; Sleep et al., 1990) and hybrid K.lactis killer toxin/MF alpha 1 (Sleep et al., 1990). The secretion leader peptides also influence the secretion efficiency and product integrity. For example, the secretion efficiency of tissue plasminogen activator in Saccharomyces cerevisiae using different leader peptides showed that, invertase signal peptide = carboxypeptidase Y signal peptide > acid phosphatase signal peptide > alpha-factor prepro > Aspergillus niger glucoamylase signal peptide (Hinnen et al., 1989). The secretory pathway provides a system for modifying and processing recombinant products, which can be employed to generate desired final product. These include disulfide bond formation, glycosylation, sequence specific endoproteolytic cleavage by subtilisin-like KEX2 protein which cleaves on the carboxyl terminal side of Lys-Arg sequence and sequence specific exoproteolytic trimming by the STE13 and KEX1 proteins (Brake et al., 1989; Hadfield et al., 1993). With regard to the promoter, it signals the initiation of transcription by RNA polymerase and the terminator signals the end. Promoters originating from yeast genes are used for heterologous gene expression, since promoters are fairly host specific and those from higher eukaryotes tend to have lower efficiency of initiation of transcription in yeast (Beggs et al., 1980; Rothstein et al., 1984; Hadfield et al., 1993). The strength of the promoter correlates with the productivity of the recombinant protein. A comparative study on the the effects of SUC2, PGK and GAL7 promoters on the secretion of alpha-amylase in Saccharomyces cerevisiae was done using identical plasmids and hosts grown under inducing conditions. It showed that the strength of the promoter (PGK > GAL7 > SUC2) correlates with the productivity (Buckholz, 1993). The twenty or so glycolytic genes of Saccharomyces cerevisiae give rise to about 50% of the cell's total cell protein. These genes are often single copy genes. It was assumed that this high-level of expression was due to the association of strong promoters with these genes. So, when strong continuous expression is desired, these promoters are used (Goodey et al., 1987). Although the expression of these genes is continuous, it is not truly constitutive, however, being induced by glucose and diminishing by a factor up to 30-fold on non-fermentable carbon sources. Some of the promoters are inducible or repressible (Brent et al., 1985), such that by the addition of an inducer or repressor, protein production is either switched on or off. This trait is very useful in achieving regulated expression of heterologous proteins in yeast (Russel et al., 1991). Generally, the inducible promoters are preferable for scale-up to long-term batch or in continuous culture, so that the growth phase may be separated from the production phase (Da Silva & Bailey, 1991). For example, the ADH2 promoter is both powerful and tightly regulated. It is repressed over 100-fold by glucose. So, this has been used to facilitate the controlled expression of a number of foreign gene products including toxic proteins (Price et al., 1990; Shuster et al., 1989). The other example, the metallothionein gene CUP1 promoter is tightly regulated and can be induced 20-fold by addition of copper ions to the medium (Etcheverry, 1990). Since copper is toxic to yeast cells, use of CUP1 promoter needs to be in conjunction with a copper-resistant cell phenotype.
Yeasts and Therapeutic Products: Problems and Prospects Although Saccharomyces cerevisiae has some limitations as a host, the majority of recombinant proteins produced in yeasts have been expressed using this host system (Table 1). Except for some notable exceptions, product yields are usually low in this host system. Even with a strong promoter, the yields of heterologous proteins reach a maximum of only 1-5% of total cellular protein. Plasmid instability is a severe problem when the product being expressed has some toxicity. Saccharomyces cerevisiae is also known to hyperglycosylate the secreted glycoproteins, with up to 100 mannose units per N-linked oligosaccharide chain (Moir et al., 1987; Lemontt et al., 1985), which may cause dramatic differences in immunogenecity, diminished activity and decreased serum retention of the recombinant protein. Given the majority of recombinant proteins for human therapy are glycosylated, it is essential to fully characterize and if possible control the degree of glycosylation of the recombinant products. Unlike Saccharomyces cerevisiae, non-Saccharomyces yeast species have glycosylation patterns closer to the mammalian high-mannose type of glycosylation (Buckholz & Gleeson, 1991). The FDA in United States and the Committee for Proprietory Medical Productions (CPMP) of the European Community are demanding increasingly sophisticated carbohydrate analysis of all glycoproteins destined for human therapy (Liu, 1992). Moreover, many Saccharomyces cerevisiae secreted proteins are not found free in the culture medium, but are retained in the periplasmic space in a cell-associated form. This can lead to problems in purification and decreased yields of purified product. In view of the above, several laboratories have looked at alternative yeast hosts for stable high-level expression and secretion of appropriately modified recombinant proteins. For example, recombinant human serum albumin (rHSA) was expressed in Saccharomyces cerevisiae at 0.6mg/L (Etcheverry et al., 1986), whereas in Kluyveromyces lactis correctly folded and processed rHSA at 300-400 mg/L level corresponding to >90% of the total secreted proteins, could be obtained in shake flask culture (Fleer et al., 1991b). The K.lactis productivity further increased to several grams/L in batch fermentation. Other example, prochymosin secretion is much more efficient from Kluyveromyces lactis and Yarrowia lipolytica than from Saccharomyces cerevisiae (Van den Berg et al., 1990; Yu, 1994). The productivity of selected recombinant therapeutic proteins expressed in different yeast hosts is shown in Table 3. Since the commercial viability of a recombinant product depends on productivity, it is apparent that the appropriate combination of gene product and yeast host needs to be investigated on an individual basis.
Conclusions Since the majority of therapeutic proteins are naturally secreted glycoproteins, in the heterologous gene expression of these proteins it is necessary that they are secreted with the correct folding and bioactivity. Given the limitations in secretion and protein modification with the E.coli host system; yeasts, being eukaryotes, have advantages over prokaryotic systems for producing recombinant proteins with post-translational modifications such as glycosylation which are essential for bioactivity. The successful production of therapeutic proteins in Saccharomyces cerevisiae with full biological activity has been demonstrated. Alternative non-Saccharomyces host systems are becoming popular for the production of recombinant proteins of medical importance. The future should see an increased use of various yeast host systems in combination with improved fermentation methodologies for the large scale production of correctly folded and bioactive recombinant therapeutic products.
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Table 1: Cloned therapeutic gene products expressed in Saccharomyces
cerevisiae
------------------------------------------------------------------------
Product type Gene product Reference
------------------------------------------------------------------------
Viral Hepatitis B surface antigen Valenzuela et al. (1982)
(HB s Ag) particles
Hepatitis B core antigen Miyanohara et al. (1986)
(HB c Ag) particles
Hybrid herpes simplex-gD: Valenzuela et al. (1985)
HBsAg particles
Hepatitis B surface antigen Miyanohara et al. (1983)
(HB s Ag)
Hepatitis B surface antigen Fu et al. (1996b)
(HB s Ag)
Hybrid foot-and-mouth disease- Beesley et al. (1990)
VPl:HB c Ag particles
Hybrid HIV-gp 120: Ty-Virus like Adams et al. (1987)
particles
Influenza viral haemagglutinin Jabbar et al. (1985)
Polio virus protein VP2 Verbakel et al. (1987)
Hormones Insulin precursors Thim et al. (1986)
Insulin Stepien et al. (1983)
Human parathoid hormone Olstad et al. (1995)
Proinsulin Cousens et al. (1987)
Hybrid human chorionic Beesley et al. (1990)
gonodotrophin: HB c Ag
Human growth hormone Tokunaga et al. (1985)
Human somatostatin Green et al. (1986)
Antibodies Synthesis and assembly of Wood et al. (1985)
functional antibodies
Functional antibody and Fab Horwitz et al. (1988)
fragments
Chorismate mutase 'abzyme'antibody Bowdish et al. (1991)
Cytokines & Granulocyte / macrophage colony- Cantrell et al. (1985)
growth stimulating factor
factors Human insulin - like growth Bayne et al. (1988)
factor 1
Human nerve growth factor Kanaya et al. (1989)
Human epidermal growth factor Brake et al. (1984)
Platelet - derived endothelial
cell growth factors Finnis et al. (1992)
Human interleukin - 1beta Baldari et al. (1987)
Human interleukin-lA Livi et al. (1991)
Human interleukin - 2 Ernst & Richman (1989)
Human interleukin - 6 Guisez et al. (1991)
Human tissue factor Stone et al. (1995)
Interferons Human leukocyte interferon- Hitzeman et al. (1981)
alpha(D)
Secretion of human interferons Hitzeman et al. (1983)
Interferon - alpha2, -beta1 and Piggot et al. (1987)
hybrid X-430
Blood Human alpha- and beta- globins Wagenbach et al. (1991)
proteins & haemoglobin
& related Human factor XIIIa Rinas et al. (1990)
products Human erythropoietin Elliot et al. (1989)
Human coagulation factor XIII Jagadeeswaran & Hass (1990)
Human serum albumin Sleep et al. (1990)
Human antithrombin III Broker et al. (1987)
Leech Hirudin Loison et al. (1988)
Hirudin Park et al. (1995)
Hirudin Sohn et al. (1995)
Human alphal-antitrypsin Cabezon et al. (1984)
Human tissue plasminogen Lemontt et al. (1985)
activator
Fibrinogen Roy et al. (1995)
Viper Echistatin (platelet Jacobson et al. (1989)
aggregation inhibitor)
Human lactoferrin Liang & Richardson (1993
Ovine beta-lactoglobulin Rocha et al. (1996)
Receptors Human oestrogen receptor Metzger et al. (1988)
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Table 2: Cloned therapeutic gene products expressed in non-Soccharomyces
yeasts
------------------------------------------------------------------------
Yeast host Gene product Reference
------------------------------------------------------------------------
Pichia pastoris Tetanus toxin fragment C Clare et al. (1991)
Human IgE receptor Basu et al. (1992)
Streptokinase Hagenson et al. (1989)
Hepatitis B surface antigert Cregg et al. (1987)
Human turnour necrosis factor Sreekrishna et al. (1989)
Ghilaten Brankamp et al. (1995)
Human proteinase inhibitor 6 Sun et al. (1995)
Hansenula polymorpha Hepatitis B surface middle Shen et al. (1989)
antigen
Hepatitis B surface S and Janowicz et al. (1991)
L antigens
Human serum albumin Hoclkins et al. (1990)
Hirudin variant HV1 Weydemann et al. (1995)
Kluyveromyces lactis Human interleukin-lB Fleer et al. (1991a)
Human serum albumin Fleer et al. (1991b)
Hepatitis B surface antigen Martinez et al. (1992)
Tissue plasminogen activator Yeh et al. (1990)
Ovine beta-lactoglobulin Rocha et al. (1996)
Schizosaccharomyces Macrophage colony stimulating Hua et al. (1994)
pombe factor (truncated)
Human antithrombin III Broker et al. (1987)
Human factor XIIIa Broker & Bauml (1989)
Yarrowia lipolitica Porcine interferon Heslot et al. (1990)
------------------------------------------------------------------------------
Table 3: Productivity of selected cloned therapeutic gene products in
different yeasts
------------------------------------------------------------------------------
Gene product Yeast host Promoter Productivity Reference
------------------------------------------------------------------------------
Human serum Saccharomyces CUP1 0.6 mg/l Etcheverry et al.(1986)
albumin cerevisiae UYP1 35-45 mg/l Sleep et al. (1990)
Kluyveromyces PGK/LAC4 300-400 mg/l Fleer et al. (1991b)
lactis
HB s Ag monomer Saccharomyces PGK1 1-2 mg/ Valenzuela et
cerevisiae 100mg protein al.(1982)
Hansenula MOX1 2.7-3.6 mg/ Shen et al. (1989)
polymorpha 100mg protein
Pichia pastoris AOXI 2.3 mg/ Cregg et al. (1987)
100mg protein
Human Saccharomyces PGK 1-2 mg/l Baldari et al.(1987)
interleukin - 1B cerevisiae
Kluyveromyces PH05 80 mg/l Fleer et al. (1991a)
lactis
Human Saccharomyces PGK 15 mg/l Tuite et al.(1982)
interferon-alpha cerevisiae
IGF-1 Saccharomyces ADH2/GAPDH 25 mg/l Shuster et al.(1989)
cerevisiae
Human lactoferrin Saccharomyces Chelatin 1.5-2 mg/l Liang & Richardson
cerevisiae (1993)
Ovine beta- Saccharomyces PGK 40-50 mg/l Rocha et al. (1996)
lactoglobulin cerevisiae
Kluyveromyces PGK 40-50 mg/l Rocha et al. (1996)
lactis
Hirudin Saccharomyces GALl0 59 mg/l Park et al. (1995)
cerevisiae
Hirudin Hansenula MOX 1-2 g/l Weydemann et al.
variant HV1 polymorpha (1995)
Fibrinogen Saccharomyces GALl 30 mg/l Roy et al. (1995)
------------------------------------------------------------------------------
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