BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution.

Autor: Pearce KH; Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA., Overton LK; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Gampe RT; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Barrett GB; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Taylor JD; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., McKee DD; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Campobasso N; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Nolte RT; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA., Reid RA; Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
Jazyk: angličtina
Zdroj: Acta crystallographica. Section F, Structural biology communications [Acta Crystallogr F Struct Biol Commun] 2019 Apr 01; Vol. 75 (Pt 4), pp. 239-245. Date of Electronic Publication: 2019 Mar 13.
DOI: 10.1107/S2053230X19001419
Abstrakt: Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
Databáze: MEDLINE
Nepřihlášeným uživatelům se plný text nezobrazuje