A rapid selective extraction procedure for the outer membrane protein (OmpF) from Escherichia coli.
Porins are essential pore-forming proteins found in the outer membrane of several gram-negative bacteria. Investigating the relationships between molecular structure and function involves an extremely time-consuming and labor-intensive purification procedure. We report a method for rapid extraction of the outer membrane protein, OmpF, from freeze-dried Escherichia coli cells using valeric acid, alleviating the effort and time in sample preparation. Extraction results in a highly enriched fraction containing OmpF as 76% of the total protein content. The apparent molecular mass determined by SDS-PAGE mobility was 38,900, similar to that of the monomeric form of OmpF. N-terminal sequencing yielded 23 amino acids with 100% identity to the published OmpF sequence. The trimeric form of OmpF was observed in unheated samples run on SDS-PAGE and analysis of these samples by periodic acid/silver staining revealed the presence of unbound lipopolysaccharides. Furthermore, this method should prove useful for isolating other outer membrane proteins.
Arcidiacono
Steven
S
U.S. Army Soldier Biological Chemical Command, Natick Soldier Center, Kansas Street, Natick, Massachusetts 01760, USA.
Butler
Michelle M
MM
Mello
Charlene M
CM
D000595:Amino Acid Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010421:Pentanoic Acids / Q000494:pharmacology; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D008070:Lipopolysaccharides; C109237:OmpF protein; D010421:Pentanoic Acids; D018272:Porins; C038780:n-pentanoic acid