Enhanced Secretion of Heterologous Proteins in Pichia pastoris Following Overexpression of Saccharomyces cerevisiae Chaperone Proteins

Zhang, Wei; Zhao, Hongfeng; Xue, Cong; Xiong, Xiang-Hua; Yao, Xue-Qin; li, xianyu; Chen, Huipeng; Liu, Zhimin

doi:10.1021/bp060019r

Cited by 85 publications

(54 citation statements)

References 14 publications

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“…As a contrasting approach, the folding and secretion apparatus of yeast can be rationally tuned by overexpression or deletion of target genes thought to play a role in protein secretion. A nonexhaustive list of examples includes the overexpression of heavy chain binding protein (BiP), yielding increased production of single-chain antibodies (scFv) (35), and scTCR (33); overexpression of PDI, increasing yields of scFv (35), granulocyte colony-stimulating factor (44), and acid phosphatase (30); and deletion of the Golgi apparatus-resident calcium ATPase gene, PMR1, increasing the production yields of prochymosin (10) and propapain (28). However, this semirational approach requires preliminary knowledge of potential gene targets.…”

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confidence: 99%

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

Wentz

Shusta

2007

Appl Environ Microbiol

113

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The yeast Saccharomyces cerevisiae is an attractive host for the production of heterologous proteins. However, low-yield production of many proteins (from micrograms to milligrams/liter) leaves considerable room for optimization. By engineering the yeast cell via traceable genome-wide libraries, genes that can enhance protein expression level because of their roles in protein transcription, translation, folding, and trafficking processes can be readily identified. This report details a novel approach that combines yeast cDNA overexpression libraries with yeast surface display to allow the rapid flow cytometric screening of engineered yeast for gene products that improve the display of heterologous proteins. After optimization of the screening conditions, a genome-wide scan yielded five yeast gene products that promoted increased display levels of a single-chain T-cell receptor (scTCR). The display-enhancing genes included those coding for cell wall proteins (CCW12, CWP2, and SED1), a ribosomal subunit protein (RPP0), and an endoplasmic reticulum-resident protein (ERO1). Under the premise that yeast surface display levels could be used as a predictor of secretion efficiency, each display-enhancing gene product was tested for its ability to affect secretion levels of multiple scTCR and single-chain antibodies (scFv). All of the selected yeast gene products were shown to promote increased secretion of active protein (1.5-fold to 7.9-fold), with CCW12 and ERO1 being the most generalizable enhancers of scFv/scTCR secretion.

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mentioning

confidence: 99%

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

Wentz

Shusta

2007

Appl Environ Microbiol

113

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“…On the other hand, Hsp70 and Hsp40 chaperone families in the cytoplasm or in endoplasmic reticulum importantly regulate the folding and the secretion of heterologous proteins [24]. As the cells entered the main reactor, they had the opportunity to recover from the stress.…”

Section: Results Doe1mentioning

confidence: 99%

Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

2013

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Pichia pastoris is an attractive candidate platform for recombinant protein production. Dissolved oxygen is one of the most important factors in the cultivation of P. pastoris. However, the effect of oxygen on triggering productivity led to ambivalent results. In our earlier work, a two-compartment system, consisting of a single reactor coupled with a plug flow reactor (PFR), has been proposed as a tool to improve protein quantity and quality. The goal of this work was to investigate the effects of frequency (the residence time of broth in the PFR) and amplitude (the dissolved oxygen level in the reactor) of the stress on productivity, titer and physiology. A recombinant P. pastoris strain, which expressed horseradish peroxidase, was used as the model system. Thirteen experiments were performed. Multivariate data analysis was done and the results showed that the residence time did not influence titer, productivity and physiology over the range of residence time studied while dissolved oxygen influenced titer and specific productivity in a quadratic function. In other words, an intermediate level of dissolved oxygen (25%) showed the highest specific productivity and titter, irrespective of the residence time in the PFR. In turn, the variation of the residence time and dissolved oxygen did not influence growth physiology, as quantified in biomass and carbon dioxide yields.

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“…Thus, it is possible and imperative to increase the secretion capacity of this important expression system, to make it more attractive and competitive. Among different technologies of increasing the secretion capacity of yeast expression host (Zhang et al, 2006), we were especially interested in the disruption of PMR1, because disruption of S. cerevisiae PMR1 increased the secretory expression level of single chain urinary plasminogen activator (scu-PA) and prochymosin by 11-and 60-fold, respectively (Harmsen et al, 1996;Melnick et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon‐α2b fusion protein

Zhao

Xue

Wang

et al. 2008

Yeast

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In an attempt to increase the secretion capacity of Pichia pastoris (Pp), PpPMR1 gene was disrupted with GS115 as parent strain, and the resultant mutant was designated as Pppmr1. Pppmr1 displayed a Ca 2+ -dependent growth defect, which was consistent with the PMR1 mutation in other yeasts. HSA-L5-IFNα2b, a human serum albumin (HSA) and inferferon-α2b (IFNα2b) fusion protein with a flexible linker of 5 amino acid residues, was employed as a reporter to study the effects of PpPMR1 disruption on the secretion of heterologous protein. Because of its decreased viability after induction, Pppmr1 secreted more HSA-L5-IFNα2b only during the early phase (the first 15 hours) of induction. Although HSA-L5-IFNα2b secreted from GS115 and Pppmr1 had similar antiviral activity, the latter was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to aggregation at neutral to mild alkaline pH). Site-directed mutagenesis revealed that the heterogeneity of HSA-L5-IFNα2b secreted from Pppmr1 was originated from the incomplete disulphide bridge pairing between Cys1 and Cys98 of IFNα2b. To be secreted homogeneously from Pppmr1 and to be stable in aqueous solution, the linker of the fusion protein should be extended to 10 amino acid residues.

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Enhanced Secretion of Heterologous Proteins in Pichia pastoris Following Overexpression of Saccharomyces cerevisiae Chaperone Proteins

Cited by 85 publications

References 14 publications

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon‐α2b fusion protein

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