Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes.

Autor: Galluccio M; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Mazza T; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Scalise M; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Tripicchio M; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Scarpelli M; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Tolomeo M; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy., Pochini L; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy.; National Research Council (CNR), Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Via Amendola 122/O, 70126 Bari, Italy., Indiveri C; Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy.; National Research Council (CNR), Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Via Amendola 122/O, 70126 Bari, Italy.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2023 Dec 30; Vol. 25 (1). Date of Electronic Publication: 2023 Dec 30.
DOI: 10.3390/ijms25010536
Abstrakt: The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C 12 E 8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na + -independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.
Databáze: MEDLINE
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