High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein
| Autor: | Juliana Lukša, Saulius Serva, Elena Servienė, Monika Podoliankaitė, Gintautas Vyšniauskis, Vytautas Melvydas, Jolanta Sereikaitė |
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| Rok vydání: | 2014 |
| Předmět: |
Saccharomyces cerevisiae
Bioengineering Protein aggregation medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Antibodies Microbiology law.invention law Gene Expression Regulation Fungal medicine Escherichia coli Animals Amino Acid Sequence Cloning Molecular Molecular Biology biology Toxin biology.organism_classification Yeast Killer Factors Yeast Polyclonal antibodies biology.protein Recombinant DNA Protein G Rabbits Biotechnology |
| Zdroj: | Molecular biotechnology. 56(7) |
| ISSN: | 1559-0305 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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