Use of bifunctional hybrid β-lactamases for epitope mapping and immunoassay development

Chevigné, Andy; Yilmaz, Nursel; Gaspard, Gilles; Giannotta, Fabrizio; François, Jean; Frère, Jean Marie; Galleni, Moreno; Filée, Patrice

doi:10.1016/j.jim.2006.12.009

Cited by 7 publications

(28 citation statements)

References 25 publications

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“…They have been selected as scaffolds to create hybrid proteins based on a series of observations. Firstly, they are fairly small (30 kDa) and stable proteins that are easily overexpressed in E. coli and subsequently purified [9–11]. Their three-dimensional structure has been determined by X-ray crystallography (Figure 1) [18, 19, 25, 26].…”

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

“…TEM-1 is a β -lactamase sensitive to many proteases [11, 29]. Some BHPs that do not confer resistance to ampicillin to E. coli (i.e., BHPs containing insertions at sites identified as non-permissive) could however be successfully expressed in protease-deficient E. coli strains.…”

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

“…Some BHPs that do not confer resistance to ampicillin to E. coli (i.e., BHPs containing insertions at sites identified as non-permissive) could however be successfully expressed in protease-deficient E. coli strains. This observation suggests that insertions at these positions into TEM-1 actually increase its sensitivity to proteolysis [11]. This increased susceptibility to proteolysis could be due to local conformational changes or to a destabilisation of the enzyme upon the insertion of the exogenous polypeptide.…”

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

“…In contrast, BlaP presents an advantage over TEM-1 in being much less sensitive to proteases. Indeed, this enzyme evolved to withstand high levels of various and numerous proteases, which are secreted by the gram-positive Bacillus licheniformis bacterium [11, 30]. Based on this observation, it was anticipated that BlaP could constitute an alternative scaffold to TEM-1 to generate hybrid proteins.…”

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

“…Such hybrid proteins can be designed for a range of applications such as (i) the creation of bifunctional hybrid proteins [8–11], (ii) the production, purification, and characterization of proteins otherwise difficult to express [10, 12, 13], (iii) the determination of the epitope of a specific antibody at the surface of an antigen [8, 11], (iv) the generation of antibodies against specific antigens or fragments thereof [9, 13–15], and (v) the investigation of fundamental aspects of structure, stability, and function of proteins [10, 12, 16]. …”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Class A -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Huynen

Filée²,

Matagne

et al. 2013

BioMed Research International

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Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications from basic research to medical applications. This review focuses on the use of class A β-lactamases as versatile scaffolds to design hybrid enzymes (referred to as β-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions. We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins.

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Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

Section: Class a β-Lactamases As Scaffolds To Create Hybrid Enzymesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Class A -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Huynen

Filée²,

Matagne

et al. 2013

BioMed Research International

Self Cite

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Comparative functional analysis of the human macrophage chitotriosidase

et al. 2011

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This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the b-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.

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

Vandevenne¹,

Gaspard²,

Yilmaz³

et al. 2008

Protein Engineering Design and Selection

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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.

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Use of bifunctional hybrid β-lactamases for epitope mapping and immunoassay development

Cited by 7 publications

References 25 publications

Class A -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Class A -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Comparative functional analysis of the human macrophage chitotriosidase

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

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