J. Bacteriol., 08 1996, 4909-4918, Vol 178, No. 16
Copyright © 1996, American Society for Microbiology

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi

JT Skare, CI Champion, TA Mirzabekov, ES Shang, DR Blanco, H Erdjument-Bromage, P Tempst, BL Kagan, JN Miller and MA Lovett
Department of Microbiology and Immunology, UCLA School of Medicine 90095, USA.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast- performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28- kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24- amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel- purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

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