Rapid and easy development of versatile tools to study protein/ligand interactions.
The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-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 beta-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.
Vandevenne
M
M
Macromolécules biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Sart-Tilman, Liège, Belgium.
Gaspard
G
G
Yilmaz
N
N
Giannotta
F
F
Frère
J M
JM
Galleni
M
M
Filée
P
P
D002176:Candida albicans / Q000378:metabolism; D002686:Chitin / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006596:Hexosaminidases / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D008024:Ligands; D002686:Chitin; D006596:Hexosaminidases; C086400:chitotriosidase; D001618:beta-Lactamases