Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores.
A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.
Saravolac
E G
EG
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Taylor
N F
NF
Benz
R
R
Hancock
R E
RE
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D018272:Porins / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008051:Lipid Bilayers; C086247:OprB protein, Pseudomonas; D018272:Porins; D005947:Glucose