Collection and characterisation of bacterial membrane proteins.
A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.
Saidijam
Massoud
M
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Psakis
Georgios
G
Clough
Joanne L
JL
Meuller
Johan
J
Suzuki
Shun'ichi
S
Hoyle
Christopher J
CJ
Palmer
Sarah L
SL
Morrison
Scott M
SM
Pos
Martin K
MK
Essenberg
Richard C
RC
Maiden
Martin C J
MC
Abu-bakr
Atif
A
Baumberg
Simon G
SG
Neyfakh
Alex A
AA
Griffith
Jeffrey K
JK
Stark
Michael J
MJ
Ward
Alison
A
O'Reilly
John
J
Rutherford
Nicholas G
NG
Phillips-Jones
Mary K
MK
Henderson
Peter J F
PJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D016480:Helicobacter pylori / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D008565:Membrane Proteins; D011994:Recombinant Proteins