Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli.
A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.
Kim
Young-Mog
YM
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.
Ogawa
Wakano
W
Tamai
Eiji
E
Kuroda
Teruo
T
Mizushima
Tohru
T
Tsuchiya
Tomofusa
T
D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry; D013696:Temperature; D013912:Threonine / Q000494:pharmacology; D013997:Time Factors
D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D008081:Liposomes; D011510:Proteolipids; C489859:SstT protein, E coli; D013912:Threonine; D006639:Histidine