title: Expressed protein ligation for a large dimeric protein.
abstract: Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic resonance (NMR) or fluorescence spectroscopy. The application for oligomeric proteins, however, is restricted by the need to obtain a large excess of active dimer over reactants and intermediates. Here, we explored the suitability of the EPL reaction for large dimeric proteins using the molecular chaperone Hsp90 as a model. We systematically varied the reaction conditions and the preparation protocols for the reactants. Modulation of the ligation site by shortening the flexible segment at the N-terminus of the C-terminal reactant increased the yield sufficiently to isolate the product by chromatography. Under those conditions, 41% of the used C-terminal fragment could be successfully ligated. We discuss possible up-scaling for segmental isotope labelling for NMR applications.
authors.affiliation: Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: G Elif
authors.initials: GE
authors.lastname: Karagöz
authors.forename: Tessa
authors.initials: T
authors.lastname: Sinnige
authors.forename: Ofey
authors.initials: O
authors.lastname: Hsieh
authors.forename: Stefan G D
authors.initials: SG
authors.lastname: Rüdiger
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization
chemical_list: D018841:HSP90 Heat-Shock Proteins