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Screening protein refolding using surface plasmon resonance.

Daniel B Jones·Matthew H Hutchinson·Anton P J Middelberg
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Abstract

Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol. The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths. One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody. This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity. The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions. SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design. The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host.

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Biosensing TechniquesDinitrochlorobenzeneEscherichia coliGlutathioneGlutathione TransferaseProtein Array AnalysisProtein BindingProtein FoldingRecombinant ProteinsSurface Plasmon ResonanceJournal ArticleResearch Support, Non-U.S. Gov't

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Daniel B Jones, Matthew H Hutchinson, Anton P J Middelberg. 2004. Screening protein refolding using surface plasmon resonance.. https://doi.org/10.1002/pmic.200300662

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