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 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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Holzenburg A,Engel A,Kessler R,Manz HJ,Lustig A,Aebi Udoi
10.1021/bi00436a010subject
Has Abstractpub_date
1989-05-16 00:00:00pages
4187-93issue
10eissn
0006-2960issn
1520-4995journal_volume
28pub_type
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