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

15 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
Publication statusPublished - 2002
Externally publishedYes

Fingerprint

Symporters
Serine
Escherichia coli
Purification
Genes
Proteins
Isopropyl Thiogalactoside
Amino Acids
Gene encoding
DNA sequences
Threonine
Codon
Liposomes
Methionine
Detergents
Dilution
Amino Acid Sequence
Membrane Proteins
Plasmids
Resins

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kim, Y. M., Ogawa, W., Tamai, E., Kuroda, T., Mizushima, T., & Tsuchiya, T. (2002). Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli. Journal of Biochemistry, 132(1), 71-76.

Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli. / Kim, Young Mog; Ogawa, Wakano; Tamai, Eiji; Kuroda, Teruo; Mizushima, Tohru; Tsuchiya, Tomofusa.

In: Journal of Biochemistry, Vol. 132, No. 1, 2002, p. 71-76.

Research output: Contribution to journalArticle

Kim, YM, Ogawa, W, Tamai, E, Kuroda, T, Mizushima, T & Tsuchiya, T 2002, 'Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli', Journal of Biochemistry, vol. 132, no. 1, pp. 71-76.
Kim YM, Ogawa W, Tamai E, Kuroda T, Mizushima T, Tsuchiya T. Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli. Journal of Biochemistry. 2002;132(1):71-76.
Kim, Young Mog ; Ogawa, Wakano ; Tamai, Eiji ; Kuroda, Teruo ; Mizushima, Tohru ; Tsuchiya, Tomofusa. / Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli. In: Journal of Biochemistry. 2002 ; Vol. 132, No. 1. pp. 71-76.
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