Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

• Published in: The Journal of biological chemistry Vol. 267; no. 8; pp. 5177 - 5183
• Main Author: URATANI, Y
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.03.1992
• Subjects: Amino Acids, Branched-Chain - metabolism; Analytical, structural and metabolic biochemistry; Bacterial Proteins; Binding and carrier proteins; Biological and medical sciences; branched-chain amino acid transporter; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell Membrane - metabolism; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Fundamental and applied biological sciences. Psychology; Immune Sera; Kinetics; Leucine - metabolism; Liposomes; Lithium - pharmacology; Molecular Weight; Proteins; Proteolipids - metabolism; Pseudomonas aeruginosa - metabolism; Sodium - pharmacology; Pseudomonadales; Proteoliposome; Purification; Sodium; Immunoaffinity chromatography; Membrane transport; Aminoacid; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa; Carrier protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42747-0

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.