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A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules Using Isothermal Titration Calorimetry

National University of Singapore

(RECEIVED May 3, 2008; ACCEPTED June 27, 2008)

The most challenging tasks in biology include the identification of (a) the orphan receptor for a ligand, (b) the ligand for an orphan receptor protein, and (c) the target protein(s) for a given drug or a lead compound which are critical for the pharmacological or side effects. At present, several approaches are available including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification or immobilization. Here we demonstrate a new universal method for fishing orphan target protein from a complex mixture of biomolecules using Isothermal Titration Calorimetry as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins; a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.

Keywords: Isolation, Characterization, Chromatography; Purification methods, general; New Methods; Calorimetry

¹ E-mail: dbsjayar{at}nus.edu.sg

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