Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes

Abstract

Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

Comparison of zonal elution and nonlinear chromatography in determination of the interaction between seven drugs and immobilised β2-adrenoceptor

“…Another notable technology in the analysis of drug-protein interactions is high performance affinity chromatography (HPAC) [19][20][21]. In HPAC, a protein is often immobilised on a solid matrix to construct an affinity stationary phase.…”

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“…To purify the protein of interest, about 300 mL of the fermentation supernatant after 144 h of methanol induction was centrifuged at 8000 × g for 5 min to remove the cells. The supernatants were collected and dialyzed against buffer A (20 mMTris-HCl; 5 mM Na 2 EDTA; pH 6.8) using a Millipore 10 kDa cut off membrane device at 4°C to remove ions and salts and then shake with Ni-NTA resin at 4°C for 3 h. After two washes with low concentrations of 25 mM imidazole, the protein was eluted with cell lysis buffer containing 10% glycerol and 300 mM imidazole to remove other proteins and small peptides as described in the Ni-NTA Resin Handbook by TransGen Biotechnology (China) (Etemadzadeh et al, 2015;Moser et al, 2014). The final, relatively pure sample was concentrated and stored in 15 mL biotin ligase storage buffer (50 mM imidazole, 50 mMNaCl, 5% glycerol, 5 mMTris-HCl, pH 6.8).…”

“…To circumvent the difficulties of confirming the activity, a sf-GFP-fused streptavidin was expressed and surface displayed on the outer membrane of E. coli strain BL21 (DE3). The streptavidin is a tetrameric molecule that have an extremely high affinity for biotin with biotin binding sites at each subunit (Moser et al, 2014;Schmitz, 2002;Sau-Ching and Sui-Lam, 2005). Sf-GFP-fused-streptavidin displayed on the cell surface can detect biotinylated protein avi-tagged mcherry.…”

Rapid analysis of interaction between six drugs and β2‐adrenergic receptor by injection amount‐dependent method

“…This makes a powerful tool for characterizing drug–protein interactions. It is remarkable that high‐performance affinity chromatography (HPAC) (Anguizola et al, ; Moser, White, & Kovacs, ; Schiel, Ohnmacht, & Hage, ) possessing new mathematical tools is relatively rapid for obtaining information on drug–protein interactions. Among the reported mathematical models, frontal analysis (Kamarei, Gritti, Guiochon, & Burchell, ; Ràfols, Zarza, & Bosch, ), zonal elution (Matsuda, Li, Zheng, & Hage, ) and nonlinear chromatography (Li et al, ) are mostly utilized to determine the binding parameters of drug–protein interactions.…”