Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli

Young Mog Kim, Wakano Ogawa, Eiji Tamai, Teruo Kuroda, Tohru Mizushima, Tomofusa Tsuchiya

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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 Histagged 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 (Ni2+-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 Km and the Vmax values for serine transport with the proteoliposomes were 0.82 μM 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°C.

Original languageEnglish
Pages (from-to)71-76
Number of pages6
JournalJournal of biochemistry
Volume132
Issue number1
DOIs
Publication statusPublished - 2002 Jul

Keywords

  • Histidine-tag
  • Purification
  • Serine
  • SstT
  • Transporter

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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