Quantitation of protein expression in a cell-free system: Efficient detection of yiel
 

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

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

J Biomol NMR. 2005 Jan;31(1):11-9

Authors: Neerathilingam M, Greene LH, Colebrooke SA, Campbell ID, Staunton D

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.

PMID: 15692735 [PubMed - indexed for MEDLINE]



Source: PubMed