title: Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.
abstract: Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.
authors.affiliation: Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Soldmannstrasse 16, 17487 Greifswald, Germany.
authors.forename: Javier F
authors.initials: JF
authors.lastname: Chaparro-Riggers
authors.forename: Roland
authors.initials: R
authors.lastname: Breves
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Uwe
authors.initials: U
authors.lastname: Bornscheuer
mesh_terms: D010447:Peptide Hydrolases / Q000737:chemistry*; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism
chemical_list: D019151:Peptide Library; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D020860:Subtilisin; C057840:omptin outer membrane protease