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Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology
Author(s) -
Szeri Flora,
Niaziorimi Fatemeh,
Donnelly Sylvia,
Orndorff Joseph,
Wetering Koen
Publication year - 2021
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13957
Subject(s) - pyrophosphate , fusion protein , chemistry , biochemistry , fluorescence , microbiology and biotechnology , intracellular , biophysics , biology , recombinant dna , enzyme , physics , gene , quantum mechanics
ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating mutations in ABCC6 give low plasma pyrophosphate and underlie the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is still unknown. Fluorescent ABCC6 fusion proteins would allow mechanistic studies, but fluorophores attached to the ABCC6 N‐ or C‐terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. A corresponding ABCC6 variant in which the catalytic glutamate of the second nucleotide binding domain was mutated, correctly routed to the plasma membrane but was inactive. Finally, N‐terminal His 10 or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization.