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

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Research Abstract Details 

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Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Abstract Text:

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

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.

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Publishing Authors By Initials

YM Kim,W Ogawa,E Tamai,T Kuroda,T Mizushima,T Tsuchiya,

For similar natural sciences: time: time factors research abstracts see: natural sciences: time: time factors research

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Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 132

Page Numbers: 71-6

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Jul

YEAR: 2002

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Keywords Mesh Terms:

KEYWORDS: Time Factors

MESH TERMS: pharmacology

Chemical & Substance for Abstract: Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli. Information

Substance Name: Threonine

Registry Number: 72-19-5

Grant and Affiliation Information for Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli.

AFFILIATION: Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

Country: Japan

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MEDLINETA: J Biochem

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