title: Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
abstract: We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of 19F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded beta-barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that 19F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK.
authors.forename: Muniasamy
authors.initials: M
authors.lastname: Neerathilingam
authors.forename: Lesley H
authors.initials: LH
authors.lastname: Greene
authors.forename: Simon A
authors.initials: SA
authors.lastname: Colebrooke
authors.forename: Iain D
authors.initials: ID
authors.lastname: Campbell
authors.forename: David
authors.initials: D
authors.lastname: Staunton
mesh_terms: D005461:Fluorine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000032:analysis*; D012177:Retinol-Binding Proteins / Q000032:analysis; D014364:Tryptophan / Q000031:analogs & derivatives*
chemical_list: D011506:Proteins; D012177:Retinol-Binding Proteins; D005461:Fluorine; C001244:6-fluorotryptophan; D014364:Tryptophan