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

Cho, Eun Young; Cheon, Seon Ah; Kim, Hyun-Ah; Choo, Jinho; Lee, Dong-Jik; Ryu, Ho Myung; Rhee, Shin Hyung; Chung, Bong-Hyun; Kim, Jeong‐Yoon; Kang, Hyun Ah

doi:10.1016/j.jbiotec.2010.06.014

Cited by 20 publications

(17 citation statements)

References 18 publications

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“…The regulation of YPS1 expression by the UPR suggests that heterologous or high-level expression could activate the UPR, leading to increased expression levels of YPS1 and, ultimately, to an increased degradation of the secreted protein. Recent results indicating that Yps2p also contributes to the degradation of some recombinant proteins (10) are also consistent with our findings that Yps1p and Yps2p both have a role in secretory pathway homeostasis.…”

Section: Discussionsupporting

confidence: 93%

“…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%

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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.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

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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“…S. cerevisiae transformants harboring the S. cerevisiae HAC1 or C. neoformans HXL1 expression vectors were selected on media lacking uracil and assayed for the sensitivity to TM and DTT. For construction of an HXL1 integration vector under the control of the S. cerevisiae HAC1 promoter, the 2.3 kb ScHAC1 promoter- HXL1 u or - HXL1 s fragments were obtained from pRE318-CnHXL1u or CnHXL1s, respectively, via treatment of NheI/PvuII and Klenow fragment, and ligated to pTcUR3 [44] treated with BamHI and Klenow fragment, generating pTcU-CnHXL1u and pTcU-CnHXL1s, respectively. These vectors were transformed into wild-type BY4742 and Schac1 strains after AvrII digestion.…”

Section: Methodsmentioning

confidence: 99%

Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of Cryptococcus neoformans

et al. 2011

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In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen Cryptococcus neoformans has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by HXL1 (HAC1 and XBP1-Like gene 1). C. neoformans HXL1 encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of HXL1 and its unconventional splicing is widely conserved in C. neoformans strains of divergent serotypes. Notably, both C. neoformans ire1 and hxl1 mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the ire1 mutant were suppressed by the spliced active form of Hxl1, supporting that HXL1 mRNA is a downstream target of Ire1. Interestingly, however, the ire1 and hxl1 mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in C. neoformans. Finally, Ire1 and Hxl1 were shown to be critical for virulence of C. neoformans, suggesting UPR signaling as a novel antifungal therapeutic target.

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“…In this case, construction of protease-deficient strains with multiple gene deletions could be employed as an efficient strategy to solve the protein cleavage problem. For example, a multiple-yapsin-deficient mutant strain of S. cerevisiae lacking the YPS1, YPS2, YPS3, YPS6, and YPS7 genes showed diminished cleavage of the recombinant parathyroid hormone protein at high cell-density cultivation during fed-batch fermentation (Cho et al, 2010). Yapsin-deficient strains have been Fig.…”

Section: Host Strain Engineering For Secretory Production Of Recombinmentioning

confidence: 99%

“…Carboxypeptidase Y Vacuolar serine-type Cterminal exopeptidase Van Den Hazel et al (1996) Sc, Pp KEX2 Proprotein convertase Serine-type calciumdependent serine protease Zhang et al (2001Zhang et al ( , 2006 and Werten & de Wolf (2005) Pp, Hp YPS1 Yapsin-1 Aspartic-type endopeptidase Werten & de Wolf (2005), Cho et al (2010) and Choi et al (2003) Sc, Kl UBI4 Ubiquitin Ubiquitin-26S proteasome system Chen et al (1994) and Bao & Fukuhara (2001) Sc, Pp, Cb…”

Section: Cpy1mentioning

confidence: 99%

Yeast synthetic biology for the production of recombinant therapeutic proteins

2014

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The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.

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

Cited by 20 publications

References 18 publications

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

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

Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of Cryptococcus neoformans

Yeast synthetic biology for the production of recombinant therapeutic proteins

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