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Development of a high-throughput cloning strategy for characterization of acinetobacter baumannii drug transporter proteins
- Publication Year :
- 2011
- Publisher :
- Switzerland : S Karger AG, 2011.
-
Abstract
- Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.
- Subjects :
- Acinetobacter baumannii
Physiology
Genetic Vectors
Biological Transport, Active
Computational biology
Microbial Sensitivity Tests
Applied Microbiology and Biotechnology
Biochemistry
Microbiology
Gene Knockout Techniques
Bacterial Proteins
Drug Resistance, Multiple, Bacterial
Escherichia coli
Humans
Vector (molecular biology)
Cloning, Molecular
MATE transporter
Expression vector
biology
Membrane transport protein
Membrane Transport Proteins
Cell Biology
biology.organism_classification
efflux
gateway cloning
Transport protein
Anti-Bacterial Agents
High-Throughput Screening Assays
Membrane protein
Biotechnology & Applied Microbiology
biology.protein
Efflux
Heterologous expression
Biotechnology
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....8466a692dfd900166f0f5215ca1961cb