Project Description

Generation of a Nuclear Receptor with known stability issues for SPR studies

Our client needed a nuclear receptor protein for Biacore Surface plasmon resonance (SPR) studies. There was precedent for the production of the protein and was available commercially, but it was well known to have stability issues.  Our challenge was to supply high quality, pure, characterised protein. The protein needed a His tag and biotinylation to allow binding to the surface of the Biacore chip and also good stability for SPR experiments to be carried out by the client.

Our Solutions

After extensive analysis of the literature and several rounds of discussion with the client, an appropriate construct was designed which fulfilled the brief regards domain boundaries and the addition of a 6His and Avi-tag to allow biotinylation. Our strategy encompassed the co-expression of the biotin ligase enzyme to allow cellular biotinylation of the Avi-tag.

The supply, and the customer’s final application, permitted the expression and purification to be carried out in the presence of a stabilising ligand, and a suitable detergent, to avoid protein aggregation and the known stability issue. Once these concerns were addressed, purification via affinity capture and a polishing size exclusion step lead to a delivery of milligram quantities of protein. The removal of the detergent was also investigated for the client with a view to supply for other screening techniques but though a second batch of protein was provided it was of much lower yield as the inherent instability returned.

The Impact

Following our strategy for expression and purification we were able to deliver protein that was >95% pure by SDS PAGE and 100% biotinylated when the intact protein was analysed by the mass spectrometry. It was also shown to have a clean, reproducible melting curve during a thermal shift QC thus giving an extra level of confidence in the quality of the protein.

Feedback from the client indicated that they were able to see a 20-fold increase in the SPR assay window in comparison to the protein material used previously.

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