title: Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors.
abstract: In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied - studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)(2)-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members.
authors.affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute, Protein Crystallography Group, Beutenbergstr. 11, D-07745 Jena, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Kuester
authors.forename: Gero L
authors.initials: GL
authors.lastname: Becker
authors.forename: Kornelia
authors.initials: K
authors.lastname: Hardes
authors.forename: Iris
authors.initials: I
authors.lastname: Lindberg
authors.forename: Torsten
authors.initials: T
authors.lastname: Steinmetzer
authors.forename: Manuel E
authors.initials: ME
authors.lastname: Than
mesh_terms: D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D045683:Furin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D011480:Protease Inhibitors / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D045683:Furin