Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

FEBS Letters

article

Flora Szeri¹  Fatemeh Niaziorimi¹  Sylvia Donnelly¹  Joseph Orndorff¹  Koen van de Wetering¹ 

[1] Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College and PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University

关键词: ABC transporter;    cellular ATP efflux;    fluorescent fusion protein;    pseudoxanthoma elasticum;    purification;   

DOI  :  10.1002/1873-3468.13957

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown