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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 C12E8 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. [ABSTRACT FROM AUTHOR]

The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to overexpress the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health. [ABSTRACT FROM AUTHOR]

Ten percent of human genes encode for membrane transport systems, which are key components in maintaining cell homeostasis. They are involved in the transport of nutrients, catabolites, vitamins, and ions, allowing the absorption and distribution of these compounds to the various body regions. In addition, roughly 60% of FDA-approved drugs interact with membrane proteins, among which are transporters, often responsible for pharmacokinetics and side effects. Defects of membrane transport systems can cause diseases; however, knowledge of the structure/function relationships of transporters is still limited. Among the expression of hosts that produce human membrane transport systems, E. coli is one of the most favorable for its low cultivation costs, fast growth, handiness, and extensive knowledge of its genetics and molecular mechanisms. However, the expression in E. coli of human membrane proteins is often toxic due to the hydrophobicity of these proteins and the diversity in structure with respect to their bacterial counterparts. Moreover, differences in codon usage between humans and bacteria hamper translation. This review summarizes the many strategies exploited to achieve the expression of human transport systems in bacteria, providing a guide to help people who want to deal with this topic. [ABSTRACT FROM AUTHOR]

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human flAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 30 -phosphoadenosine 50 -phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of flAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L-1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min-1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6h of induction with IPTG at 28°C. The expressed protein with an apparent molecular mass of 54kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6h of induction with IPTG at 28°C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3M urea and applied to a Ni2+-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50mM imidazole, 0.1% Triton X-100 and 50mM 2-mercaptoethanol. A yield of about 3mg purified protein per liter of cell culture was obtained. [Copyright &y& Elsevier]

Abstract: The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni2+-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [3H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na+. Internal Na+ slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [3H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [3H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na+ on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism. [Copyright &y& Elsevier]