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High-Yield Expression in Escherichia coli,Purification and Application of Budding Yeast K2 Killer Protein
Authors:Monika Podoliankaitė  Juliana Lukša  Gintautas Vyšniauskis  Jolanta Sereikaitė  Vytautas Melvydas  Saulius Serva  Elena Servienė
Institution:1. Laboratory of Genetics, Institute of Botany, Nature Research Centre, Akademijos Str. 2, 08412, Vilnius, Lithuania
2. Department of Biopharmacy, State Research Institute for Innovative Medicine, Zygimantu 9, 01102, Vilnius, Lithuania
3. Department of Chemistry and Bioengineering, Faculty of Fundamental Sciences, Vilnius Gediminas Technical University, Sauletekio 11, 10223, Vilnius, Lithuania
4. Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Vilnius University, Ciurlionio 21, 03101, Vilnius, Lithuania
Abstract:Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.
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