Expression of Recombinant Proteins in Yeast

February 9, 2006
Daniel Rudolph, Ph.D.

,
Don R. Durham, Ph.D.

,
Aaron Heifetz, PhD

,
Sriram Srinivasan, Ph.D.

BioPharm International, BioPharm International-02-09-2006, Volume 2006 Supplement, Issue 1

Yeast systems have been a staple for producing large amounts of proteins for industrial and biopharmaceutical use for many years. Yeast can be grown to very high cell mass densities in well-defined medium. Recombinant proteins in yeast can be over-expressed so the product is secreted from the cell and available for recovery in the fermentation solution. Proteins secreted by yeasts are heavily glycosylated at consensus glycosylation sites. Thus, expression of recombinant proteins in yeast systems historically has been confined to proteins where post-translations glycosylation patterns do not affect the function of proteins. Several yeast expression systems are used for recombinant protein expression, including Sacharomyces, Scizosacchromyces pombe, Pichia pastoris and Hansanuela polymorpha.

Yeast systems have been a staple for producing large amounts of proteins for industrial and biopharmaceutical use for many years. Yeast can be grown to very high cell mass densities in well-defined medium. Recombinant proteins in yeast can be over-expressed so the product is secreted from the cell and available for recovery in the fermentation solution. Proteins secreted by yeasts are heavily glycosylated at consensus glycosylation sites. Thus, expression of recombinant proteins in yeast systems historically has been confined to proteins where post-translations glycosylation patterns do not affect the function of proteins. Several yeast expression systems are used for recombinant protein expression, including Sacharomyces, Scizosacchromyces pombe, Pichia pastoris and Hansanuela polymorpha.

In this article,* we discuss some of the technologies and aspects associated with biomanufacturing of therapeutics in yeast systems and highlight some techniques that are used in either basic research or clinical manufacturing, but are not typically utilized for both.

Yeast Expression Vectors

The expression of human proteins in prokaryotes has limitations in that prokaryotes do not have compartmentalized secretion system like eukaryotes and hence post-translational modifications like glycosylation do not occur. In cases like antibodies, these limiations also affect the proper folding of the proteins and hence their applicability. As a result, these proteins have to be made in higher eukaryotic mammalian cell systems. But mammalian systems are expensive and may render a very low yield. As a result, alternative systems using lower eukaryotes like yeasts Saccharomycescerevisiae, Schizosaccharomyces pombe, and methylotropic yeasts like Pichia pastoris, and Pichia methanolica have gained importance. These systems do not glycosylate the same way as that of human glycosylation. For example, S. cerevisiae produces high mannose structures, and has been useful in producing properly folded active and soluble multi-subunit proteins.

As in any prokaryote, yeast expression systems also require an origin of replication or integration, a strong promoter, and a selection marker. Expression of recombinant proteins in S. cerevisiae can be done using three types of vectors: integration vectors (YIp), episomal plasmids (YEp), and centromeric plasmids (YCp).

YIp Vectors. The YIp integrative vectors are vectors that do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector sequence flanked by two direct copies of the yeast sequence. Typically, YIp vectors integrate as a single copy. However, methods to integrate multiple copies and stable cell lines with up to 15-20 copies of recombinant gene integrations have been developed for over-expressing specific genes.2 YIp plasmids with two yeast segments, such as YFG1 and the URA3 marker, have the potential to integrate at either of the genomic loci, whereas vectors containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of the multiple sites within genome.2

YEp Vectors. The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 μm plasmid that serves as an origin of replication (2 μm ori). The 2 μm ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. Most YEp plasmids are relatively unstable and even under conditions of selective growth, only 60 to 95 percent of the cells retain the YEp plasmid. The copy number of most YEp plasmids ranges from 10 to 40 copies per cell. Although this system is used for small scale expression studies, the use of YEp vectors in large-scale manufacturing is not advisable.

YCp Vectors. YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences (CEN), and autonomously replicating sequences (ARS). The YCp vectors are typically present at very low copy numbers from 1 to 3 per cell. These vectors are also relatively unstable and not very useful in high level expression but are used as regular cloning vectors (e.g., pYC2, pBM272).

Selection Markers

Yeast selection markers can be classified into two types: complementation markers and dominant selection markers. Dominant selection markers are antibiotic markers that can be used in yeast such as G418 and cyclohexamide. Complementation markers are marker genes that complement an auxotrophic mutation in the genome like URA3, TRPI, HIS3, and LEU2. These auxotrophic markers are used in selection of recombinants with all three types of expression systems (integration, episomal, and centromeric plasmids). A summary of the various plasmids and their selection systems is shown in Table 1.

Table 1. Saccharomyces Plasmids.

Promoters

A range of yeast promoters is available for protein expression. Some like ADH2,SUC2 are inducible and others like GAPDH are constitutive in expression. Similar to the selection markers, a wide variety of combinations of promoters, markers, and expression systems are commercially available (Table 2).

Table 2. Saccharomyces Promoters.

Secretion Signals

A variety of secretion signals are also available for expression in S. cerevisiae. These include:

  • Prepro alpha factor12

  • HSp15012

  • PHO112

  • SUC212

  • KILM1 (killer toxin type 1)12

  • GGP113

These systems and combinations of promoter, vectors, and signal sequence can be used for high-level expression of recombinant proteins in yeast.

Pichia Pastoris

Pichia pastoris, a methyltrophic yeast that grows to high cell density in a defined medium, is capable of a high level of recombinant gene expression from a tightly controlled promoter, and capable of several post-translational modifications performed by higher eukaryotes.14 Alcohol oxidase (AOX) catalyzes the first step in metabolism of methanol, and because alcohol oxidase has a low affinity for O2, the yeast expresses large amounts of alcohol oxidase, up to 30% of total cell protein, to compensate.14,15 Thus, it was determined that utilization of this promoter in the presence of methanol could lead to high recombinant protein production. The glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter has since been developed for constitutive expression of non-toxic proteins in Pichia.16 The promoter for the Pichia glutathione-dependent formaldehyde dehydrogenase (fld) gene is also available as an inducible promoter with induction by methyl-amine.17 Higgins and Cregg list numerous proteins that have been produced in Pichia.14

Recombinant genes are transferred to Pichia pastoris by shuttle vectors designed with the promoter, a cloning site for gene insertion, regulatory elements for maintenance in E. coli, and regulatory elements for maintenance and expression in Pichia. The vector is integrated into the Pichia genome by homologous recombination, either at the promoter site (AOX or GAP), or at the site of an auxotrophic gene (i.e., His4 in the commonly used GS115 strain). Thus, integration of a plasmid would allow selection on media devoid of a given amino acid such as histadine. Alternatively, vectors are available with the Sh ble gene, allowing for selection in media containing Zeocin. Zeocin is toxic and should be avoided in the manufacture of clinical use materials. Secretion signals are available for Pichia that will allow accumulation of recombinant proteins in culture medium with the signal sequences removed during secretion. The secretion signal from the Saccharomyces α-factor is the most common, but the signal sequence of the acid phosphatase (PHO1) gene is also available.14

The site of insertion of a recombinant gene will determine the phenotype of an otherwise wild-type recombinant Pichia strain. A vector cloned into a selection gene, such as His4, will grow normally on methanol, a Mut+ phenotype. There are two genes that encode AOX: AOX1, from which most AOX is derived, and AOX2, from which about 15% of AOX is derived.15 The AOX1 promoter is used to drive recombinant expression. If the vector is integrated into the host AOX1 site, the ability of the strain to utilize methanol is significantly diminished, leading to a slow methanol utilization or MutS phenotype. If both the AOX1 and AOX2 genes are knocked out, then the strain cannot utilize methanol and has a Mut phenotype. The non-wild type strains will sometimes express higher levels of product and have different fermentation strategies that can simplify the process.14,18

Stratton et al. provide methods for growth and induction of product from each phenotype and advantages for each.18 An important note is that Pichia growth is rapid on glycerol or glucose, but these carbon sources repress expression from the AOX1 promoter. The primary advantage of the Mut+ strain is the rapid growth on methanol and high rate of production of foreign protein. The disadvantage is sensitivity to high methanol concentrations and a need to regulate the amount of methanol fed, typically requiring biomass accumulation using glycerol or glucose and a subsequent shift to methanol. The MutS strains are much less sensitive to methanol concentration but grow much more slowly on methanol. Mut strains cannot utilize methanol as a carbon source, and another source is needed, but can use methanol as an inducer. Thus, feeding a limiting amount of carbon source and maintaining a low level of methanol will allow for culture growth and product production. A side benefit of the use of lower amounts of methanol is related to the safety aspects of methanol, which is flammable and combustible. Large-scale fermentation will require a significant amount of methanol and proper measures must be in place to ensure safety. A license from Research Corporation Technologies is necessary for the use of Pichia pastoris as a production strain for commercial or research purposes.

Recently, a novel system with the capability of producing recombinant glycoproteins in yeast has emerged with glycosylation sequences similar to secreted human glycoproteins produced in mammalian cells. The glycosylation pathway of Pichia pastoris was modified by eliminating endogenous enzymes, which add high mannose chains to N-glycosylation intermediates. In addition, at least five active enzymes, involved in synthesizing humanized oligosaccharide chains, were specifically transferred into P. pastoris. The results by Choi, et al. and by Hamilton et al. demonstrated the proper localization of mannosidases I and II, N-acetylglucosaminyltransferases I and II, and UDP-GlcNAc transporter into recombinant strains of P. pastoris lacking the Och1p alpha-1-6-mannosyltransferase, which initiates yeast but not human type glycosylation.19,20

The ability to produce large quantities of humanized glycoproteins in yeast could offer advantages in that glycosylated structures could be highly uniform and easily purified. In addition, cross-contamination with mammalian viruses and other mammalian host glycoproteins may be eliminated by using fed-batch production in yeast with much shorter fermentation times than mammalian cells.

Other Yeast Systems

A few other fungal systems have also been successfully used for recombinant protein production. Some of these systems are based on non-fermentation yeast species like Pichia methanolica and Hansenula polymorpha. These are similar to Pichia pastoris and can be grown to very high cell densities and high-level protein expression has been achieved. There is also a wide range of expression systems similar to the Pichia and Saccharomyces system available for recombinant protein expression. Table 3 shows some available expression technologies for these yeast systems.

Table 3. Other yeast expression systems

This article is an excerpt from the chapter, "Advances in Improved Expression of Recombinant Proteins in Microbial Systems," in Eric S. Langer, Ed., Advances in Large-Scale Biopharmaceutical Manufacturing and Scale-Up Production (ASM Press, 2004).

Daniel Rudolph, Ph.D., is a process engineer in the process development group of Cambrex Bio Science Baltimore, Inc., 5901 E. Lombard Street, Baltimore, MD 21224, 410.563.9200, Daniel.Rudolph@cambrex.com. Co-authors Sriram Srinivasan, Ph.D., Don R. Durham, Ph.D., and Aaron Heifetz, Ph.D., previously served at Cambrex in the roles of process engineer, upstream process development group; vice president of technology; and vice president, general manager, and site director, respectively.

References

1. Rudolph D, Srinivasan S, Durham DR, Heifetz A. Advances in improved ex-pression of recombinant proteins in microbial systems. In: Langer ES, editor. Advances in large-scale biopharm-aceutical manufacturing and scale-up production. Volume 1: emerging tech-nologies and scientific advancements. Washington DC: ASM Press; 2004 October. p. 194-228.

2. Parekh RN, Shaw MR, Wittrup KD. An integrating vector for tunable, high copy, stable integration into the 1996dis-persed Ty delta sites of Saccharomyces cerevisiae. Biotechnol. Prog. 1996; 12(1):16-21.

3 Nasmyth KA, Reed SL. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA 1980; 77(4):2119-2123.

4 Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 1981;78(10):6354-6358.

5 Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell 1982; 28(1): 145-154.

6 Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 1987; 60(2-3):237-243.

7 Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989; 122(1): 19-27.

8 Law RHP, Rowley MJ, Mackay IR, Corner B. Expression in Saccharomyces cerevisiae of antigenically and enzy-matically active recombinant glutamuic acid decarboxylase. J. of Biotechnology 1998; 61:57-68.

9 Bitter GA, Egan KM. Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter. Gene. 1984; 32(3):263-74.

10 Dumont J, Legrain M, Portetelle D, Brasseur R, Burny A, Hilger F. High yield synthesis of the bovine leukemia virus (BLV) p24 major internal protein in Saccharomyces cerevisiae. Gene 1989; 79(2):219-26.

11 Nishizawa M, Ozawa F, Hishinuma F. Construction of an expression and secretion vector for the yeast Saccharomyces cerevisiae. Agric. Biol. Chem. 1987; 51:515-521.

12 Chaudhuri B, Steube K, Stephan C. The pro-region of the yeast prepro-alpha-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo. European J. of Biochem. 1992; 206(3):793-800.

13 Pignatelli R, Vai M, Alberghina L, Popolo L. Expression and secretion of beta-galactosidase in Saccharomyces cerevisiae using the signal sequences of GgpI, the major yeast glycosylphosphati-dylinositol-containing protein. Biotechnol. Appl. Biochem. 1998; 27(Pt 2):81-8.

14 Higgins DR, Cregg JM. Introduction to Pichia pastoris. In: Higgins DR and Cregg JM eds. Pichia protocols, methods in molecular biology. Vol. 103. Totowa NJ: Humana Press, 1998.

15 Trinh LB, Phue JN, Shiloach J. Effect of methanol feeding strategies on recombinant mouse endostatin. Biotechnology and Bioengineering 2003; 82(4):438-444.

16 Vassileva A, Chugh DA, Swaminathan S, Khanna N. Expression of hepatitis B surface antigen in the methylotropic yeast Pichia pastoris using the GAP promoter. J. of Biotechnology 2001; 88:21-35.

17 Shen S, Sulter G, Jeffries TW, Cregg JM. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia Pastoris. Gene 1998; 216(1):93-102.

18 Stratton J, Chiruvolu V, Meagher M. High cell-density fermentation. In: Higgins DR and Cregg JM eds. Pichia protocols. op cit.

19 Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA. 2003; 100(9):5022-5027.

20 Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, et al. Production of complex human glycoproteins in yeast. Science 2003; 301(5637):1244-1246.

21 Ferminan E, Dominguez A. Heterologous protein secretion directed by a repressible acid phosphatase system of Kluyveromyces lactis: characterization of upstream region-activating sequences in the KIPHO5 gene. Appl. Environ. Microbiol. 1998; 64(7): 2403-2408.