title: The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal.
abstract: Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, 480 Ray C. Hunt Drive, Charlottesville, VA 22908, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Vergis
authors.forename: Michael C
authors.initials: MC
authors.lastname: Wiener
mesh_terms: D002846:Chromatography, Affinity; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases