Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.
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.
Chaparro-Riggers
Javier F
JF
Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Soldmannstrasse 16, 17487 Greifswald, Germany.
Breves
Roland
R
Maurer
Karl-Heinz
KH
Bornscheuer
Uwe
U
D010447:Peptide Hydrolases / Q000737:chemistry*; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism
D019151:Peptide Library; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D020860:Subtilisin; C057840:omptin outer membrane protease