Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
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.
Neerathilingam
Muniasamy
M
Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK.
Greene
Lesley H
LH
Colebrooke
Simon A
SA
Campbell
Iain D
ID
Staunton
David
D
D005461:Fluorine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000032:analysis*; D012177:Retinol-Binding Proteins / Q000032:analysis; D014364:Tryptophan / Q000031:analogs & derivatives*
D011506:Proteins; D012177:Retinol-Binding Proteins; D005461:Fluorine; C001244:6-fluorotryptophan; D014364:Tryptophan