Disruption of theSaccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin

Kerry-Williams, S.M.; Gilbert, Sarah C.; Evans, Leslie; Ballance, D. J.

doi:10.1002/(sici)1097-0061(19980130)14:2<161::aid-yea208>3.0.co;2-y

Cited by 41 publications

(5 citation statements)

References 24 publications

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“…It is because of this selective specificity that a broad range of heterologously expressed proteins in S. cerevisiae are cleaved in a limited way by these enzymes (25,26). To circumvent this problem, researchers have utilized yeast mutants that lack either yapsin 1 (27)(28)(29) or both yapsin 1 and 2 (25,30) to express their proteins. However, although recovery of intact protein increases in this double mutant, the overall yield may be limited because this mutant exhibits impaired growth at 37°C (5).…”

Section: Fig 8 Analysis Of Purified Of Sap9pmentioning

confidence: 99%

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

Cawley

Chino

Maldonado

et al. 2003

Journal of Biological Chemistry

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The potent peptidic inhibitor, Y1, of the basic residuespecific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin The apparent K i of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nM, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as ␣ and ␤, similar to yapsin 1.

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Section: Fig 8 Analysis Of Purified Of Sap9pmentioning

confidence: 99%

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

Cawley

Chino

Maldonado

et al. 2003

Journal of Biological Chemistry

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“…For example, the deletion of extracellular protease Ski5p increased the secretion of killing toxin by approximately 10-fold [111]. The Yap3p protease as an important factor in the degradation of secreted heterologous proteins, its disruption generated a significant increase in products quality including recombinant human albumin (rHA) and rHA-human growth hormone fusion protein (rHA-hGH) [112].…”

Section: Vesicle Trafficking Engineeringmentioning

confidence: 99%

Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

Zhao,

Ma,

Zhang

et al. 2024

Microb Cell Fact

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Microbial proteins are promising substitutes for animal- and plant-based proteins. S. cerevisiae, a generally recognized as safe (GRAS) microorganism, has been frequently employed to generate heterologous proteins. However, constructing a universal yeast chassis for efficient protein production is still a challenge due to the varying properties of different proteins. With progress in synthetic biology, a multitude of molecular biology tools and metabolic engineering strategies have been employed to alleviate these issues. This review first analyses the advantages of protein production by S. cerevisiae. The most recent advances in improving heterologous protein yield are summarized and discussed in terms of protein hyperexpression systems, protein secretion engineering, glycosylation pathway engineering and systems metabolic engineering. Furthermore, the prospects for efficient and sustainable heterologous protein production by S. cerevisiae are also provided.

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“…Yapsin 1 (Yps1p), a member of the yapsin family, plays an important role in degrading recombinant protein in yeast ( Kerry-Williams et al, 1998 ). Yps1p is a glycosylphosphatidylinositol (GPI)-anchored aspartic peptidases with an optimum pH of 4.0–5.0, and can recognize not only paired basic residues but also a monobasic residue accompanied by an additional basic amino acid at a specific upstream or downstream position ( Yao et al, 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris

et al. 2022

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IntroductionPichia pastoris is widely used for the production of recombinant proteins, but the low production efficiency hinders its wide application in biopharmaceuticals. Moreover, many biopharmaceutical-like proteins are accompanied by degradation during secretory expression in P. pastoris.ObjectiveIn this study, we used human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) fusion protein as a model protein to investigate whether YPS1 and YPT7 gene disruption and N-acetyl-L-cysteine (NAC) supplementation have synergistic effects to inhibit the degradation of recombinant proteins.Results and discussionOur results showed that YPS1 gene disruption reduced the degradation of intact HSA-pFSHβ and increased the yield of intact protein in the culture medium and cells without affecting the integrity of the cell wall. Moreover, the beneficial effects of YPS1 gene disruption were associated with the upregulation of the MAPK signaling pathway and maintenance of redox homeostasis. YPS1 gene disruption and NAC supplementation had synergistic effects on HSA-pFSHβ production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSHβ protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSHβ strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSHβ proteins, and synergistically increase the yield of intact HSA-pFSHβ with NAC supplementation. This study provided a valuable reference for reducing recombinant protein degradation and therefore improving the yield of recombinant proteins in P. pastoris.

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Disruption of theSaccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin

Cited by 41 publications

References 24 publications

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris

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