Abstract
The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actinobacteria / genetics
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Actinobacteria / metabolism*
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Amino Acid Sequence
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Animals
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Biological Transport / drug effects
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism
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Cloning, Molecular
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Consensus Sequence
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DNA Primers
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Membrane Glycoproteins / chemistry
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Membrane Glycoproteins / metabolism
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Membrane Transport Proteins / chemistry
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Membrane Transport Proteins / drug effects
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Membrane Transport Proteins / genetics
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Membrane Transport Proteins / metabolism*
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Molecular Sequence Data
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Nerve Tissue Proteins*
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Promoter Regions, Genetic
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Rats
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Serotonin / metabolism*
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Serotonin Plasma Membrane Transport Proteins
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Sodium / pharmacology
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Tryptophan / metabolism*
Substances
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Carrier Proteins
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DNA Primers
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Membrane Glycoproteins
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Membrane Transport Proteins
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Nerve Tissue Proteins
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Recombinant Proteins
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Serotonin Plasma Membrane Transport Proteins
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Slc6a4 protein, rat
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Serotonin
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Tryptophan
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Sodium