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- Title
Bacterial over-production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands.
- Authors
Galluccio, Michele; Tripicchio, Martina; Console, Lara; Indiveri, Cesare
- Abstract
Background: SLC38A2 is a ubiquitously expressed Na+-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy. Methods and Results: The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane. Conclusions: The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.
- Subjects
LIGANDS (Biochemistry); TYPE 2 diabetes; OVERPRODUCTION; AFFINITY chromatography; GENETIC vectors; LIGAND binding (Biochemistry)
- Publication
Molecular Biology Reports, 2024, Vol 51, Issue 1, p1
- ISSN
0301-4851
- Publication type
Article
- DOI
10.1007/s11033-023-08976-3