1. Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast
- Author
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Zahid Mushtaq, Alexander Shekhtman, Andrew Gould, Krishnappa Jagadish, Subhabrata Majumder, Julio A. Camarero, and Radhika Borra
- Subjects
Models, Molecular ,Phenotypic screening ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Cyclotides ,Protein Engineering ,Catalysis ,Article ,law.invention ,Trans-Splicing ,Plasmid ,Transformation, Genetic ,law ,Humans ,Amino Acid Sequence ,biology ,Chemistry ,General Medicine ,General Chemistry ,Protein engineering ,biology.organism_classification ,Yeast ,Recombinant Proteins ,Cyclotide ,Biochemistry ,Recombinant DNA ,alpha-Synuclein - Abstract
We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells. more...
- Published
- 2015