Methods, compositions, and kits for protein crystallization

Abstract

The present invention provides methods, compositions, and kits for protein crystallization. The present invention involves electrophoretically focusing at least a first protein species within a matrix comprising at least 2 regions of different pH, the protein being present in amount sufficient to permit crystallization within said pH gradient.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provisional patent application Ser. No. 60 / 525,811 filed Nov. 28, 2003, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION [0002] Knowledge of the three dimensional structure of a protein is often necessary in order fully to elucidate its biological function and its interaction with other proteins, and with non-protein cofactors, in a physiological pathway. Knowledge of a protein's 3D structure is critical to the rational design of small chemical entities capable of interacting with the protein with high affinity, a process used both for creation and for optimization of pharmaceutical compounds intended for ultimate use in the clinic. [0003] Although solution phase NMR spectroscopy has shown promise in elucidating protein 3D structure, its use is circumscribed by various technical limitations, and X-ray crystallography remains the principal ...

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Benefits of technology

[0009] Without intending to be bound by theory, it would appear that as a protein approaches its isoelectric point and thereby becomes more concentrated, the loss of net charge reduces its aqueous solubility, facilitating crystallization. Thus, applied to the gradient in sufficient amount, a protein may be observed to crystallize at or near its isoelectric focal point, particularly when solubilizing and denaturing agents that are typically used in analytical isoelectric focusing (IEF) are omitted during the electrophoretic procedure.

[0013] The method is simple, rapid, applicable to a wide variety of proteins, including proteins in an inhomogeneous admixture, and requires minimal user intervention, thus providing significant advantages over methods currently used in the art.

Problems solved by technology

Although solution phase NMR spectroscopy has shown promise in elucidating protein 3D structure, its use is circumscribed by various technical limitations, and X-ray crystallography remains the principal means by which three dimensional protein structures are determined at the atomic level.

Yet crystallization of proteins remains an inexact and cumbersome art.

Even when a positive result is obtained in the form of a crystal, preparing and mounting the crystal on an x-ray machine can be an arduous, time-consuming process that often results in crystal degradation or even loss.

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