A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance

Abstract

Surface plasmon resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high-affinity interactions such as biotin/streptavidin or antibody/antigen. In this chapter we describe a robust method to covalently immobilize His(6) fusion proteins on the sensor surface for SPR analysis.

Figure 1. Identification of Left and Right pump inlets on Biacore 3000

Loosen the two screws (white circles/arrows) on the panel to remove the cover. This allows the user to trace each inlet back to the right and left pump. The Biacore Running Buffer should be placed in the inlet corresponding to the left pump while the right pumps inlet should be submerged in Dispenser Buffer.

Figure 2. Loading – A typical Sensogram generated during the production of a single flow cell using the capture coupling method

The flow cell surface was stabilized by the injection of regeneration buffer (20 μl Regeneration Buffer at 20 μl/min) followed by binding of nickel to the surface (40 μl Nickel Sulfate Solution at 20 μl/min). The surface was then activated for primary amine coupling by the injection of coupling solution (30 μl EDC/NHS at 5 μl/min). His₆-Gα fusion protein in pH 7.4 Running Buffer was then injected over the surface (66 μl of 1 μM His₆-Gα at 5 μl/min). To block uncoupled primary amines on the sensor surface, ethanolamine was injected over the surface (35 μl ethanolamine at 5 μl/min). To prevent further immobilization of His₆-fusion proteins and to remove non-covalently coupled His₆-Gα, EDTA-containing Regeneration buffer was injected (20 μl at 20 μl/min).

Figure 3. Curves – Capture coupling was used to generate a sensor surface with a novel His₆-RGS protein

Four flow cells were prepared: FC1 (“REGEN FC”) - a blank surface that had 20 μl of Regeneration Buffer injected over it; FC2 (“BLANK FC”) - a non-treated NTA surface that only had experimental injections; FC3 (“His₆-Gα FC”) - ~6000 RUs of His₆-Gα were immobilized; FC4- ~6000 RUs of a novel His₆-RGS protein. Varying concentrations of His₆-Gα were injected over all four flow cells, in the presence of Transition State Running Buffer, using the KINJECT command (300 μl injections with a 200 second dissociation phase at 20 μl/min). Specific binding of the injected Gα was only seen to His₆-RGS as would be expected (2); however, the qualitative appearance of the curves changed depending on the flow cell used to subtract non-specific binding and changes in the refractive index upon injection of buffers. (A) Specific binding was determined by subtracting non-specific binding to flow cell 1 (REGEN FC) that had 20 μl of regeneration buffer injected over it prior to injections. (B) Non-specific binding was subtracted by using a flow cell loaded with a non-interacting protein immobilized on the surface (i.e., Gα does not dimerize). (C) An otherwise untreated flow cell (BLANK FC) was used to subtract non-specific binding and changes in refractive index. (D) Using equilibrium saturation analysis, dissociation constants (Kd values) for the Gα/RGS protein-protein interaction were determined to be 469 (95% C.I. 426–512) nM, 218 (95% C.I. 149–288) nM, and 651 (95% C.I. 389–917) nM using background subtraction to a regenerated blank flow cell, an immobilized His₆-Gα flow cell, and a blank flow cell, respectively.