title: Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases.
abstract: Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional multi-step chromatography from native or recombinant tag-free sources. Alternatively, they can be expressed and purified using a cleavable N-terminal affinity tag that is subsequently removed by a site-specific protease. Proteolytic tag-removal can also be performed "on-column". We show here that this not only represents a very efficient workflow, but also drastically improves the purity of the resulting protein preparations. Precondition for effective on-column-cleavage is, however, that the tag-cleaving protease does not bind the stationary phase. We introduce scAtg4 and xlUsp2 as very good and bdSENP1, bdNEDP1 as well as ssNEDP1 as ideal proteases for on-column cleavage at 4°C. Four of these proteases (bdSENP1, bdNEDP1, scAtg4, xlUsp2) as well as TEV protease display orthogonal, i.e. mutually exclusive cleavage specificities. We combined these features into a streamlined method for the production of highly pure protein complexes: Orthogonal affinity tags and protease recognitions modules are fused to individual subunits. Following co-expression or in-vitro complex assembly, consecutive cycles of affinity capture and proteolytic release then select sequentially for the presence of each orthogonally tagged subunit, yielding protein complexes of well-defined subunit stoichiometry.
authors.affiliation: Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany. Electronic address: sfrey@gwdg.de.
authors.forename: Steffen
authors.initials: S
authors.lastname: Frey
authors.affiliation: Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany. Electronic address: goerlich@mpibpc.mpg.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Görlich
mesh_terms: D002846:Chromatography, Affinity; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025842:SUMO-1 Protein / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D006639:Histidine; D010447:Peptide Hydrolases
keywords: Affinity tag; Controlled subunit stoichiometry; On-column cleavage; Protein complex; Tag-removing protease