Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Xue, Yuan; Zhao, Hong; Xue, Cong; Zhang, Wei; Xiong, Xiang Hua; Wang, Zhi Wei; Li, Xian Yu; Liu, Zhi Min

doi:10.1016/j.jbiotec.2008.09.006

Cited by 27 publications

(28 citation statements)

References 21 publications

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“…Several recent studies have reported the presence of yapsin members in other fungi (Gagnon-Arsenault et al, 2006) and have shown that Yps1p is responsible for the aberrant proteolytic cleavage of various heterologous proteins secreted from other nonSaccharomyces yeast, such as Pichia pastoris (Yao et al, 2009) and Ogata minuta (Kuroda et al, 2007). It would be interesting to investigate the presence of multiple members of yapsin genes in other industrial yeast species in order to develop multiple-yapsindeficient strains as useful host strains with reduced aberrant proteolytic cleavage activity.…”

Section: Discussionmentioning

confidence: 99%

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae

Cho

Cheon

Kim

et al. 2010

Journal of Biotechnology

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Section: Discussionmentioning

confidence: 99%

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae

Cho

Cheon

Kim

et al. 2010

Journal of Biotechnology

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“…PEP4 encodes a major vacuolar aspartyl protease which is able to activate itself as well as further proteases such as carboxypeptidase Y ( PRC1 ) and proteinase B ( PRB1 ). The use of protease-deficient strains other than the above mentioned (e.g., yps1 , kex1 , kex2 ) was reported with variable success (Ni et al 2008; Werten and de Wolf 2005; Wu et al 2013; Yao et al 2009). A general conclusion from these studies is that in many cases several proteases are involved in degradation events and, therefore, it is not an easy task to optimize protein expression by knocking out just a single one.…”

Section: Host Strain Developmentmentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

807

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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“…A number of groups have found that the presence of yapsins and their homologues adversely affects the production of heterologously expressed, recombinant proteins in S. cerevisiae and Pichia pastoris (5,10,29,41) by causing a large amount of degraded protein. Our data implicating yapsins in secretory pathway quality control provide a compelling physiological explanation for these observations.…”

Section: Discussionmentioning

confidence: 99%

“…The yapsins are the founding members of a family of GPI-linked yeast aspartyl proteases that are present in many fungi, including pathogenic yeasts such as Candida albicans (1) and Candida glabrata (18) and industrially important yeasts such as Pichia pastoris (41). Although the physiological roles of the yapsins are just beginning to be understood, this family of proteases appears to function as secretases that release membrane and cell wall-localized proteins from the cell surface (14,15,20,38).…”

mentioning

confidence: 99%

Extracellular Secretion of Overexpressed Glycosylphosphatidylinositol-Linked Cell Wall Protein Utr2/Crh2p as a Novel Protein Quality Control Mechanism in Saccharomyces cerevisiae

Miller¹,

DiDone

Krysan

2010

Eukaryot Cell

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Eukaryotic cells employ a variety of mechanisms to maintain protein quality control and homeostasis. Here we provide evidence that one such mechanism in Saccharomyces cerevisiae involves the regulated release of excess or misfolded proteins to the extracellular space. The overexpression of an epitope-tagged allele of the glycosylphosphatidylinositol (GPI)-linked cell wall protein Utr2/Crh2p (Utr2/Crh2-green fluorescent protein [GFP] or -hemagglutinin [HA]) causes endoplasmic reticulum (ER) stress and the secretion of Crh2-GFP/HA into the extracellular space. Secretion is dependent on two GPI-linked aspartyl proteases (Yps1p/2p) and components of the unfolded protein response (Ire1p and Hac1p) but is independent of ER-associated degradation (ERAD) components such as Hrd1p and Doa10p. Supporting the idea that this process represents a mechanism for protein quality control, the level of Crh2-HA is increased in strains lacking Bst1p, a protein required for the proteasomal degradation of GPI-linked proteins. Furthermore, secretion is dependent on Sec18p, indicating that it requires ER-to-Golgi trafficking, and accordingly, Crh2-HA accumulates in the ER in ire1⌬ and bst1⌬ mutants by cycloheximide chase experiments. Since a fraction of Utr2/Crh2-GFP properly localizes to the cell wall in an ire1⌬ mutant, extracellular secretion appears to occur through a pathway that is distinct from the normal GPI protein-trafficking pathway. Taken together, these data support a model in which the unfolded protein response (UPR)/yapsin-mediated extracellular release of overexpressed Utr2/ Crh2-HA or -GFP is an alternative pathway for the removal of excess or misfolded secretory proteins functioning in parallel with proteasome-mediated degradation in S. cerevisiae. This model provides an explanation for the deleterious effects of Yps1/2p on the industrial production of some recombinant proteins in S. cerevisiae.

show abstract

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Cited by 27 publications

References 21 publications

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Extracellular Secretion of Overexpressed Glycosylphosphatidylinositol-Linked Cell Wall Protein Utr2/Crh2p as a Novel Protein Quality Control Mechanism in Saccharomyces cerevisiae

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