Rapid isolation of OmpF porin-LPS complexes suitable for structure-function studies
- 1 May 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (10) , 4187-4193
- https://doi.org/10.1021/bi00436a010
Abstract
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 s2o,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.This publication has 2 references indexed in Scilit:
- Porin channels in Escherichia coli: studies with beta-lactams in intact cellsJournal of Bacteriology, 1983
- Recognition Site for Phosphorus‐Containing Compounds and Other Negatively Charged Solutes on the PhoE Protein Pore of the Outer Membrane of Escherichia coli K12European Journal of Biochemistry, 1982