Annals of Microbiology, 57 (4) 553-560 (2007) The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris Yewang ZHANG1*, Ruijiang LIU1, Xiaoyu WU2 1Department of Pharmaceutical Engineering, School of Pharmaceutics, Jiangsu University, Zhenjiang, 212013; 2School of Biochemical Engineering, Jiangxi Agricultural University, Nanchang, 330045, P.R. China Received 12 April 2007 / Accepted 25 September 2007 Abstract - The Pichia pastoris expression system has been successfully used for production of various recombinant heterogeneous proteins. The productivity of P. pastoris can be improved substantially by bioreactor cultivations. However, heterologous proteins degradation increases as well in high-cell density culture. Proteolytic degradation is a serious problem since the yeast has been employed to express recombinant proteins. In this review, some of the recent developments, as well as strategies for reducing pro- teolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels on the cytosolic proteasome, vac- uolar proteases, and proteases located within the secretory pathway in P. pastoris, are reviewed. Key words: Pichia pastoris, heterologous protein, degradation, proteases, proteasome. INTRODUCTION Some strategies have been used to overcome the proteol- ysis, including selection of protease deficient host strains The methylotrophic yeast, Pichia pastoris, is widely used as (Gleeson et al., 1998; Goodrick et al., 2001), modification host strain for the production of a variety of heterologous of the protein structure to resist the protease (Gustavsson proteins (Cregg et al., 2000). There are several factors that et al., 2001), addition of protease inhibitors in bioreactor account for its popularity, such as the strong and highly (Holmquist et al., 1997), changing the pH of the culture efficient alcohol oxidase (AOX) promoter, the stable inte- medium (Kang et al., 2000; Jahic et al., 2003a), supple- gration of expression plasmids at specific sites in P. pastoris mentation of the medium with casamino acids and peptone genome, the availability of host and vector as a commer- (Clare et al., 1991b) and addition of protease inhibitors cial kit and the practical techniques for high-cell density (Shi et al., 2003), have been suggested as means to culture. The productivity of P. pastoris in shake-flasks is reduce proteolytic degradation of the recombinant protein. normally low but can be improved greatly by bioreactor cul- However, no in-depth analysis on the conditions that pro- tivations (Cregg et al., 2000). Unfortunately, the concen- mote proteolysis or the nature of the proteases acting on tration of other cellular materials, such as proteases, the desired protein is exactly known. The review, here, increases as well in high-cell density culture. These mate- introduced the proteases found in P. pastoris and these rials can greatly reduce the desire protein production, recent developments, as well as strategies for reducing mainly due to proteolysis of recombinant proteins in P. pas- proteolytic degradation of the expressed recombinant pro- toris bioreactor cultures (Clare et al., 1991b; Brierley, tein on cultivation, cellular and molecular levels. 1998). Proteolytic degradation has been a perpetual prob- lem since yeast has been employed to express recombinant proteins (Van Den Hazel et al., 1996). Yeast vacuoles con- PROTEASES IN Pichia pastoris tain various proteases whose levels vary according to the nutritional conditions. Saccharomyces cerevisiae is known Since the proteases of yeast that were first identified and to express several proteases, some of which have been well characterised were detected biochemically and proved to identified (Van Den Hazel et al., 1996). In comparison, the be of vacuolar (lysosomal) origin, an additional nonvacuo- proteases in P. pastoris are not well characterised, although lar set was defined genetically by mutations that caused there have been several reports of proteolytic degradation incomplete proteolytic processing of precursors to killer of recombinant proteins produced in P. pastoris (Werten et toxin and the pheromone α-factor (Jones, 1991). al., 1999; Zhou and Zhang, 2002; Sinha et al., 2003). Intracellular proteases in yeast have been studied wide- ly in the last decades and they were found have vital impor- * Corresponding author. Phone: (86)5118791564; Fax: (86) tance in the desired protein production at cellular lever. A 5115038451-806; E-mail: [email protected] lot of researches were carried out on Saccharomyces cere- 554 Y. Zhang et al. visiae, and numerous proteolytic enzymes have been found precursor of 405 amino acids. A hydrophobic signal peptide and their biochemical and genetical characterisation have is found at the N-terminus of the precursor, and this pep- been studied (Van Den Hazel et al., 1996; Flores et al., tide is cleaved off upon translocation into the lumen of the 1999). The same protease system occurs in P. pastoris. endoplasmic reticulum (ER) (Klionsky et al., 1988). This There are mainly three groups of proteases in yeast: glycoprotein of 42 kDa carries two asparagine-linked gly- cytosolic proteosome, vacuolar proteases and proteases cosyl side chains; its four cysteines are thought to form located along with the secretory pathway (Jones, 1991). disulfide bonds. Three proteolytic cleavages occur during the posttranslational maturation of Pep4p. After removal of Cytosolic proteasome the signal sequence by signal peptidase, another 47 amino Achstetter et al. (1984) reported purification of a large pep- acids are removed from the NH, terminus late in the Golgi tidase, which was proved to contain 10-12 no identical sub- or in the vacuole. This intramolecular reaction is autocat- units and showed homology to the 20S cylindrical particles alytic, since mutational change of either aspartate residue in many eukaryotic cells. The peptidases were named pro- of the active site (Asn ➝ Asn) results in nearly complete some, proteasome, multiprotease complex and multicat- failure of processing (van den Hazel et al., 1995). The final alytic protease, respectively. Proteasomes are large multi- cleavage removes 7 amino acids, it is catalysed by PrB catalytic protease complexes, which fulfil central functions (Klionsky et al., 1988). in major intracellular proteolytic pathways of the eukaryot- ic cell. The 20S proteasome, which was found in the cyto- Proteinase B plasm and the nuclear of all eukaryote, is 700 kDa cylindri- The vacuolar endoprotease proteinase B (PrB), encoded by cally shaped particle. The proteasome is composed of 14 the PRB1 gene, is a member of the subtilisin family of ser- different subunits (MW 22-25 kDa), which are arranged in ine proteases (Moehle et al., 1987). The mature enzyme a stack of 4 rings with 7-fold symmetry. The 26S protea- has a molecular mass of about 31-33 kDa. Cloning and somes are an even larger proteinase complexes (about sequencing of the structural gene forPrB (PrB1) revealed a 1700 kDa), which degrade ubiquitinylated proteins in an surprisingly large open reading frame, coding for a precur- ATP-dependent fashion in vitro. The 26S proteasomes are sor of at least 69 kDa (Moehle et al., 1987). This initial pre- built up from the 20S proteasome, work as core particle cursor is translocated into the ER. Subsequently, the signal with two additional 19S complexes on both ends of the 20S peptide of about 20 amino acids and a large N-terminal cylinder. Proteasomes are needed in stress-dependent and propeptide of about 260 amino acids are removed, yielding ubiquitin-mediated proteolysis (Hilt and Wolf, 1995). The a smaller precursor, proPrB(40-42 kDa). The removal of the cytosolic proteasome (multicatalytic proteinase system) is N-terminal propeptide is presumably autocatalytic, as it responsible for the selection and rapid degradation of does not occur in strains producing an active-site mutant short-lived proteins and proteins that are detrimental to the form of PrB. This step occurs before delivery to the Golgi cell growth. The interplay between vacuolar and proteo- complex since it is not dependent on a functional Sec l 8p some proteolysis is a key regulatory cell process, particu- (Moehle et al., 1989; Hirsch et al., 1992). The large initial larly in response to stress (Hilt and Wolf, 1992; Van Den PrB precursor contains five acceptor sites for Asn-linked Hazel et al., 1996). glycosylation. However, glycosylation occurs only at one site in the precursor, and the mature protein only contains Vacuolar proteases O-linked glycosylation. The large N-terminal propeptide is Vacuolar proteases are responsible for a large fraction of required for correct biosynthesis of PrB, preventing N-linked the total cellular proteolysis in yeast, particularly under glycosylation at other sites (Nebes and Jones, 1991). The conditions of nutrient deprivation. The vacuolar uptake large initial PrB precursor is transiently active in the ER and mechanisms of the proteins destined for degradation had, removes the N-terminal propeptide autocatalytically. This until recently, received little attention. These mechanisms, yields the smaller precursor, proPrB, which is subsequently which include autophagocytosis and endocytosis, have inhibited by the non-covalently linked large propeptide. been characterised (Van Den Hazel et al., 1996). The vac- Upon arrival of this complex in the vacuole, the large uolar proteases are involved in processes of cell differenti- propeptide is