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 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 to 5×104. This demonstrates the potential for using yeast to perform phenotypic screening of genetically-encoded cyclotide-based libraries in eukaryotic cells.
Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (about 28-37 amino acids long) that share a disulfide-stabilized core (three disulfide bonds) characterized by an unusual knotted arrangement. [1] Cyclotides contrast with other circular polypeptides in that they have a well-defined three-dimensional structure, and despite their small size can be considered as microproteins. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation (Scheme 1).Furthermore, their well-defined structures have been associated with a wide range of biological functions.[2, 3] Cyclotides MCoTI-I/II are powerful trypsin inhibitors (K i % 20-30 pm) that have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of the cucurbitaceae family.[4] Although MCoTI cyclotides do not share significant sequence homology with other cyclotides beyond the presence of the three cystine bridges, structural analysis by NMR spectroscopy has shown that they adopt a similar backbone-cyclic cystine-knot topology. [5,6] MCoTI cyclotides, however, show high sequence homology with related cystineknot squash trypsin inhibitors, [4] and therefore represent interesting molecular scaffolds for drug design.
Summary
Cyclotides are fascinating micro-proteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and interconnecting peptide backbones, forming what is called a cystine knot topology. Naturally-occurring cyclotides have shown to posses various pharmacologically-relevant activities, and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.
Fremdling im Ring: Die Expression von Cyclotiden mit nichtnatürlichen Aminosäuren (grauer Kreis im Schema) in lebenden Bakterienzellen gelingt mithilfe eines hoch effizienten getrennten Inteins in Kombination mit Nonsense‐Codon‐Suppressor‐tRNA‐Technik. p‐Azidophenylalanin‐haltige Cyclotide können in Bakterien exprimiert und leicht über kupferfreie Klick‐Reaktionen markiert werden, um Cyclotid‐Protein‐Wechselwirkungen nachzuvollziehen.
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 ahighly 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 a-synuclein in live yeast cells.C yclotide MCoCP4 was selected by phenotypic screening from cells transformed with am ixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in ar atio of 1:510 4 .T his demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.
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