Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.

Journal article

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: Neerathilingam, M; Greene, L; Colebrooke, SA; Campbell, I; Staunton, D

• Publication status: Published
• Journal: Journal of biomolecular NMR
• Volume: 31
• Issue: 1
• Pages: 11-19
• Publication date: 2005-01-01
• DOI: 10.1007/s10858-004-5357-6
• EISSN: 1573-5001
• ISSN: 0925-2738
• Language: English
• Keywords: Humans; Magnetic Resonance Spectroscopy; Retinol-Binding Proteins; Tryptophan; Fluorine; Proteins