Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.
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.
Skare
J T
JT
Department of Microbiology and Immunology, UCLA School of Medicine 90095, USA.
Champion
C I
CI
Mirzabekov
T A
TA
Shang
E S
ES
Blanco
D R
DR
Erdjument-Bromage
H
H
Tempst
P
P
Kagan
B L
BL
Miller
J N
JN
Lovett
M A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D015748:Borrelia burgdorferi Group / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007473:Ion Channels / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018272:Porins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D007473:Ion Channels; D010446:Peptide Fragments; D018272:Porins; D011994:Recombinant Proteins; C100895:protein Oms28, Borrelia burgdorferi