{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T05:09:10Z","timestamp":1773292150832,"version":"3.50.1"},"reference-count":36,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2006,4,7]],"date-time":"2006-04-07T00:00:00Z","timestamp":1144368000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2006,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background:<\/jats:title><jats:p>Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods:<\/jats:title><jats:p>We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere\u2010based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCyt\u00ae flow cytometry system.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results:<\/jats:title><jats:p>We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition of the proteases, and demonstrated high throughput detection of these inhibitors.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions:<\/jats:title><jats:p>The assay developed here is adaptable to any site\u2010specific protease, compatible with high throughput flow cytometry systems, and multiplexable. Coupled with flow cytometry, which provides continuous time resolution and intrinsic resolution of free vs. bound fluorophores, this assay will be useful for high throughput screening of protease inhibitors in general and could simplify assays designed to determine protease mechanism. \u00a9 2006 International Society for Analytical Cytology<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.a.20268","type":"journal-article","created":{"date-parts":[[2006,4,7]],"date-time":"2006-04-07T22:47:10Z","timestamp":1144450030000},"page":"342-352","source":"Crossref","is-referenced-by-count":17,"title":["Microsphere\u2010based protease assays and screening application for lethal factor and factor Xa"],"prefix":"10.1002","volume":"69A","author":[{"given":"Matthew J.","family":"Saunders","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heungbok","family":"Kim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Travis A.","family":"Woods","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John P.","family":"Nolan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Larry A.","family":"Sklar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce S.","family":"Edwards","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven W.","family":"Graves","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2006,4,18]]},"reference":[{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0009-8981(99)00228-4"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.280.5364.734"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.1998.9040"},{"key":"e_1_2_6_5_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M211262200"},{"key":"e_1_2_6_6_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj3520739"},{"key":"e_1_2_6_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/0928-4257(96)80550-X"},{"key":"e_1_2_6_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2004.04.011"},{"key":"e_1_2_6_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.062171599"},{"key":"e_1_2_6_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/418386a"},{"key":"e_1_2_6_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0003-2697(03)00424-X"},{"key":"e_1_2_6_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2005.03.013"},{"key":"e_1_2_6_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt973"},{"key":"e_1_2_6_14_2","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb711"},{"key":"e_1_2_6_15_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja0495359"},{"key":"e_1_2_6_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1359-6446(01)01793-7"},{"key":"e_1_2_6_17_2","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1093\/clinchem\/27.6.816","article-title":"Automation of plasma antithrombin III assays","volume":"27","author":"Holmes EW","year":"1981","journal-title":"Clin Chem"},{"key":"e_1_2_6_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0003-2697(02)00009-X"},{"key":"e_1_2_6_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/1359-6446(96)10026-X"},{"key":"e_1_2_6_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0003-2670(02)01436-8"},{"key":"e_1_2_6_21_2","doi-asserted-by":"publisher","DOI":"10.1006\/abio.2000.4539"},{"key":"e_1_2_6_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1367-5931(00)00099-5"},{"key":"e_1_2_6_23_2","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0320(19961201)25:4<374::AID-CYTO9>3.0.CO;2-D"},{"key":"e_1_2_6_24_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biophys.31.082901.134406"},{"key":"e_1_2_6_25_2","doi-asserted-by":"publisher","DOI":"10.1002\/1097-0320(20010501)44:1<83::AID-CYTO1085>3.0.CO;2-O"},{"key":"e_1_2_6_26_2","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.10035"},{"key":"e_1_2_6_27_2","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.10124"},{"key":"e_1_2_6_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(89)90602-7"},{"key":"e_1_2_6_29_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.112220599"},{"key":"e_1_2_6_30_2","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.20138"},{"key":"e_1_2_6_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0091-679X(01)63018-8"},{"key":"e_1_2_6_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1046-5928(03)00132-3"},{"key":"e_1_2_6_33_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.49.33064"},{"key":"e_1_2_6_34_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M306466200"},{"key":"e_1_2_6_35_2","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.68.3.373-402.2004"},{"key":"e_1_2_6_36_2","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.10056"},{"key":"e_1_2_6_37_2","doi-asserted-by":"publisher","DOI":"10.1002\/1097-0320(20010101)43:1<23::AID-CYTO1015>3.0.CO;2-D"}],"container-title":["Cytometry Part A"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fcyto.a.20268","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.a.20268","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,12]],"date-time":"2023-09-12T03:42:48Z","timestamp":1694490168000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.a.20268"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2006,4,18]]},"references-count":36,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2006,5]]}},"alternative-id":["10.1002\/cyto.a.20268"],"URL":"https:\/\/doi.org\/10.1002\/cyto.a.20268","archive":["Portico"],"relation":{},"ISSN":["1552-4922","1552-4930"],"issn-type":[{"value":"1552-4922","type":"print"},{"value":"1552-4930","type":"electronic"}],"subject":[],"published":{"date-parts":[[2006,4,18]]}}}