Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding...

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni²⁺-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 10⁵ M^-1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni²⁺-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 10⁷, 1.91 × 10⁶, 2.25 × 10⁷, and 2.43 × 10⁷ M^-1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 10⁷, 4.52 × 10⁶, 1.20 × 10⁷, and 1.56 × 10⁷ M^-1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.