scholarly journals Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

2005 ◽  
Vol 71 (5) ◽  
pp. 2310-2317 ◽  
Author(s):  
Marc W. T. Werten ◽  
Frits A. de Wolf
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Pichia Pastoris ◽  
Single Gene ◽  
Foreign Protein ◽  
Heterologous Proteins ◽  
Basic Residue ◽  
Substrate Specificities ◽  
Basic Amino Acids ◽  
High Level

ABSTRACT Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae α-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the α-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the α-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

scholarly journals Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

2004 ◽  
Vol 70 (2) ◽  
pp. 961-966 ◽  
Author(s):  
Antje Eiden-Plach ◽  
Tatjana Zagorc ◽  
Tanja Heintel ◽  
Yvonne Carius ◽  
Frank Breinig ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Schizosaccharomyces Pombe ◽  
Protein Secretion ◽  
Candida Glabrata ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Foreign Protein ◽  
Green Fluorescent

ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Expression, Purification and Characterization of Recombinant Human Gelatin in Pichia pastoris

2011 ◽  
Vol 236-238 ◽  
pp. 2905-2912 ◽  
Author(s):  
Bin Liu ◽  
Yun Ting Lei ◽  
Jing Zhang ◽  
Li Hu ◽  
Shu Lin Yang
Keyword(s):  
Amino Acids ◽  
Pichia Pastoris ◽  
Recombinant Protein Expression ◽  
Uv Spectroscopy ◽  
Gel Filtration ◽  
Type Iii Collagen ◽  
High Level Expression ◽  
Secreted Expression ◽  
High Level ◽  
Expression Strain

Based on the idiographic character of collagenous domain of human type III collagen, a recombinant human gelatin monomeric gene (gel) was designed and synthesized. All hydrophobic amino acids (proline excluded) were replaced by hydrophilic amino acids to improve the hydrophilic properties, and the codons encoding amino acids were optimized according to Pichia pastoris bias usage. Then a recombinant human gelatin expression vector pPIC9KG6 containing six monomeric genes ligated in the same orientation was constructed successfully. After verificated the validity of construction by DNA sequencing, the recombinant vector pPIC9KG6 was electroporated into the Pichia pastoris GS115, and Mut+ pPIC9KG6 transformants were selected on the basis of G418 resistance. Then a high-level expression strain was picked up from transformants by analyzing their recombinant protein expression levels. SDS-PAGE analysis of cell lysate and fermentation supernatant of the high-level expression strain showed that recombinant human gelatin can be expressed intracellularly and secreted expression, and its expression level reaches 16.06 g per liter. Secreted recombinant human gelatin was purified from fermentation supernatant by gel filtration chromatography. By UV spectroscopy and FTIR and SEM, it was confirmed that purified recombinant human gelatin is similar to animal-derived gelatin in protein structure.

Ygr125w/Vsb1-dependent accumulation of basic amino acids into vacuoles of Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Miyuki Kawano-Kawada ◽  
Haruka Ichimura ◽  
Shota Ohnishi ◽  
Yusuke Yamamoto ◽  
Yumi Kawasaki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transmembrane Helix ◽  
Membrane Vesicles ◽  
Vacuolar Membrane ◽  
Expression Plasmid ◽  
Basic Amino Acids ◽  
Uptake Activity ◽  
Hydrophilic Region ◽  
Aspartate Residue

Abstract The Ygr125w was previously identified as a vacuolar membrane protein by a proteomic analysis. We found that vacuolar levels of basic amino acids drastically decreased in ygr125wΔ cells. Since N- or C-terminally tagged Ygr125w was not functional, an expression plasmid of YGR125w with HA3-tag inserted in its N-terminal hydrophilic region was constructed. Introduction of this plasmid into ygr125w∆ cells restored the vacuolar levels of basic amino acids. We successfully detected the uptake activity of arginine by the vacuolar membrane vesicles depending on HA3-YGR125w expression. A conserved aspartate residue in the predicted first transmembrane helix (D223) was indispensable for the accumulation of basic amino acids. YGR125w has been recently reported as a gene involved in vacuolar storage of arginine; and it is designated as VSB1. Taken together, our findings indicate that Ygr125w/Vsb1 contributes to the uptake of arginine into vacuoles and vacuolar compartmentalization of basic amino acids.

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