title: An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins.
abstract: Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species.
authors.affiliation: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Katsiaryna
authors.initials: K
authors.lastname: Tarasava
authors.affiliation: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland freisinger@chem.uzh.ch.
authors.forename: Eva
authors.initials: E
authors.lastname: Freisinger
mesh_terms: D000595:Amino Acid Sequence; D003500:Cyclization; D003545:Cysteine / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017800:Potyvirus / Q000737:chemistry / Q000201:enzymology; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D014908:Triticum
chemical_list: D010456:Peptides, Cyclic; D010940:Plant Proteins; D011994:Recombinant Proteins; D008668:Metallothionein; D010450:Endopeptidases; C099488:TEV protease; D003545:Cysteine
keywords: TEV protease; cyclic proteins; cysteine; intein tags; metallothionein