A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Citations Over TimeTop 10% of 2010 papers

Abstract

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

Related Papers

• → Maltose-Binding Protein Fusion Allows for High Level Bacterial Expression and Purification of Bioactive Mammalian Cytokine Derivatives(2014)15 cited
• → Production of extracellular domain of human tissue factor using maltose-binding protein fusion system(2002)11 cited
• → Production of the chimeric-binding protein, maltose-binding protein-protein a, by gene fusion(1995)8 cited
• Expression and Identification of the Fusion of GnRH-6 with the Gene Coding for Maltose Binding Protein(2007)
• Expression and Purification of Secreted form TNF-related Activation-induced Cytokine in E.coli as a Maltose Binding Protein Fusion(2001)