Optimized production and analysis of the staphylococcal multidrug efflux protein QacA.
The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression/purification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.
Hassan
Karl A
KA
School of Biological Sciences, University of Sydney, Sydney, NSW, Australia.
Xu
Zhiqiang
Z
Watkins
Ryan E
RE
Brennan
Richard G
RG
Skurray
Ronald A
RA
Brown
Melissa H
MH
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002942:Circular Dichroism; D003062:Codon / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013210:Staphylococcus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003062:Codon; D026901:Membrane Transport Proteins; C068498:qacA protein, Staphylococcus aureus