Rapid isolation of OmpF porin-LPS complexes suitable for structure-function studies.
A gentle and rapid isolation procedure is described yielding fractions containing better than 95% pure OmpF porin of Escherichia coli BE with different amounts of bound lipopolysaccharide (LPS). The procedure employs continuous free-flow electrophoresis (FFE) in the presence of detergent above its critical micelle concentration. Total yields of around 45% were typically obtained when porin-enriched membrane extracts were processed. By use of analytical ultracentrifugation a molecular mass of 114,000 and a sedimentation coefficient S20,w of 5.0 S were determined for porin trimers containing approximately 1 mol of tightly bound LPS. This porin readily formed 3D crystals suitable for high-resolution X-ray diffraction analysis. Three other porin-LPS isoforms isolated by FFE revealed molecular masses of 120,000, 124,000, and 151,000, suggesting that, in addition to the tightly bound LPS, 1, 2, and 8 mol of loosely bound LPS were present per mole of porin trimer. Each of the four different isoforms was suitable for reconstitution into highly ordered protein-lipid membrane arrays. The membrane crystals obtained with the 151-kDa isoform exhibited a unit cell polymorphism similar to that previously reported.
Holzenburg
A
A
Maurice E. Müller Institute for High Resolution Electron Microscopy, Basel, Switzerland.
Engel
A
A
Kessler
R
R
Manz
H J
HJ
Lustig
A
A
Aebi
U
U
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D003460:Crystallization; D004586:Electrophoresis; D004926:Escherichia coli / Q000032:analysis; D008070:Lipopolysaccharides / Q000302:isolation & purification*; D008970:Molecular Weight; D018272:Porins; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides; D018272:Porins