Seeking connectivity between engineered proteins and transducers: connection for glutathione S-transferase fusion proteins on surface plasmon resonance devices Original Research Article

The affinity bond between glutathione (GSH) and glutathione S-transferase (GST) is exploited as a means for ‘connectivity’ for engineered proteins at a surface plasmon resonance (SPR) surface. If the protein of interest is recombinantly fused to GST the resulting fusion protein can be linked specifically to GSH self-assembled on a gold surface. Classical self-assembly and the potential assisted self-assembly were compared. The classical method produced unstable layers. Applying a potential during the assembly process significantly improved stability and reproducibility. Suitable GSH layers could be deposited at potentials >0.2 V versus Ag/AgCl, where the reductive desorption showed no desorption peaks between −0.85 and −1.1 V. The GSH-functionalised surface was tested for applicability with the plant cyclin-dependent kinase (CDK) Cdc2aAm recombinantly fused to GST. The fusion protein maintained both the affinity for glutathione and the activity for cyclin binding of its parent proteins. SPR signals due to the interaction of Cdc2aAm with a cyclin-binding-site specific antibody were confirmed by ELISA. In this instance, this test system opens up the possibility of studying the cell cycle machinery, but more widely the issues concerned with maintaining the correct conformations of proteins to achieve protein arrays can be developed from this method.