title: Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast.
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.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA).
authors.forename: Krishnappa
authors.initials: K
authors.lastname: Jagadish
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA).
authors.forename: Andrew
authors.initials: A
authors.lastname: Gould
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA).
authors.forename: Radhika
authors.initials: R
authors.lastname: Borra
authors.affiliation: Department of Chemistry, State University of New York, Albany, NY 12222 (USA).
authors.forename: Subhabrata
authors.initials: S
authors.lastname: Majumder
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA).
authors.forename: Zahid
authors.initials: Z
authors.lastname: Mushtaq
authors.affiliation: Department of Chemistry, State University of New York, Albany, NY 12222 (USA).
authors.forename: Alexander
authors.initials: A
authors.lastname: Shekhtman
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA). jcamarer@usc.edu.|Department of Chemistry, University of Southern California, Los Angeles, CA 90033 (USA). jcamarer@usc.edu.
authors.forename: Julio A
authors.initials: JA
authors.lastname: Camarero
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D020040:Trans-Splicing; D014170:Transformation, Genetic; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D047169:Cyclotides; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051844:alpha-Synuclein
keywords: aptamers; biological activity; peptides; protein expression; synuclein