Large-scale preparation of bacterial cell membranes by tangential flow filtration.
The preparation of cell membranes by ultracentrifugation of bacterial cell lysates, a pre-requisite for the purification of over-expressed membrane proteins, is both time-consuming and difficult to perform on a large scale. To overcome this bottleneck in the structural investigation of such proteins in the UK Membrane Protein Structure Initiative, we have investigated the alternative use of tangential flow filtration for preparation of membranes from Escherichia coli. This method proved to be superior to the conventional use of ultracentrifuges both in speed and in yield of membrane protein. Moreover, it could more readily be scaled up to process larger quantities of bacterial cells. Comparison of the purity and monodispersity of an over-expressed membrane protein purified from conventionally-prepared membranes and from membranes prepared by filtration revealed no substantial differences. The approach described should therefore be of general use for membrane protein preparation for a wide range of applications, including both structural and functional studies.
Roach
Peter C J
PC
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Postis
Vincent L G
VL
Deacon
Sarah E
SE
Wright
Gareth S A
GS
Ingram
Jean C
JC
Xia
Xiaobing
X
McPherson
Michael J
MJ
Baldwin
Stephen A
SA
D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000737:chemistry / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D008851:Micropore Filters; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000096:biosynthesis / Q000302:isolation & purification; D014461:Ultracentrifugation
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III