PEDS Advance Access originally published online on May 2, 2008
Protein Engineering Design and Selection 2008 21(7):443-451; doi:10.1093/protein/gzn021
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Rapid and easy development of versatile tools to study protein/ligand interactions
1Macromolécules biologiques, Centre d’Ingénierie des Protéines 2ProGenosis 3Laboratoire d’Enzymologie, Centre d’Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium
4 To whom correspondence should be addressed. E-mail: pfilee{at}ulg.ac.be
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.
Keywords: β-lactamase/antibodies/high through put screening/hybrid protein
Received December 19, 2007; revised April 2, 2008; accepted April 2, 2008.