Rapid and easy development of versatile tools to study protein/ligand interactions

Abstract

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the β-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid β-lactamases is achieved in the presence of β-lactams making further screening of correctly folded and secreted hybrid β-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the β-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the β-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules. © The Author 2008. Published by Oxford University Press. All rights reserved.