Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.
The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.
Uratani
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Y
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D007106:Immune Sera; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008081:Liposomes; D008094:Lithium / Q000494:pharmacology; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D002352:Carrier Proteins; D007106:Immune Sera; D008081:Liposomes; D011510:Proteolipids; C028332:leucine-isoleucine-valine binding protein, bacteria; C020485:proteoliposomes; D008094:Lithium; D012964:Sodium; D007930:Leucine