Characterization of a Functional Bacterial Homologue of Sodium-dependent Neurotransmitter Transporters

By using a T7 promoter-based system, TnaT with an N-terminal His6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield, and transport was Na+-dependent and highly selective.

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.