A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily.
A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)(6)-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.
Szakonyi
Gerda
G
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, Institute for Membrane and Systems Biology, University of Leeds, Leeds, UK.
Leng
Dong
D
Ma
Pikyee
P
Bettaney
Kim E
KE
Saidijam
Massoud
M
Ward
Alison
A
Zibaei
Saeid
S
Gardiner
Alastair T
AT
Cogdell
Richard J
RJ
Butaye
Patrick
P
Kolsto
Anne-Brit
AB
O'reilly
John
J
Hope
Ryan J
RJ
Rutherford
Nicholas G
NG
Hoyle
Christopher J
CJ
Henderson
Peter J F
PJ
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D005798:Genes, Bacterial / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D018435:ATP Binding Cassette Transporter, Subfamily B; D000900:Anti-Bacterial Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D006639:Histidine; C024504:bicozamycin