{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T13:27:25Z","timestamp":1758634045874},"reference-count":49,"publisher":"American Society for Microbiology","issue":"23","license":[{"start":{"date-parts":[[2014,12,1]],"date-time":"2014-12-01T00:00:00Z","timestamp":1417392000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2014,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:named-content content-type=\"genus-species\">Clostridium botulinum<\/jats:named-content>\n            subtype A4 neurotoxin (BoNT\/A4) is naturally expressed in the dual-toxin-producing\n            <jats:named-content content-type=\"genus-species\">C. botulinum<\/jats:named-content>\n            strain 657Ba at 100\u00d7 lower titers than BoNT\/B. In this study, we describe purification of recombinant BoNT\/A4 (rBoNT\/A4) expressed in a nonsporulating and nontoxigenic\n            <jats:named-content content-type=\"genus-species\">C. botulinum<\/jats:named-content>\n            expression host strain. The rBoNT\/A4 copurified with nontoxic toxin complex components provided in\n            <jats:italic>trans<\/jats:italic>\n            by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT\/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT\/A1, and the recombinant BoNT\/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT\/A4 light chain (LC) expressed in\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            has indicated reduced stability and activity of BoNT\/A4 LC compared to BoNT\/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT\/A4, a recombinant full-length BoNT\/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the\n            <jats:italic>in vitro<\/jats:italic>\n            , cellular, and\n            <jats:italic>in vivo<\/jats:italic>\n            activities of rBoNT\/A4 and rBoNT\/A4-L260F I264R showed 1,000-fold-lower activity than BoNT\/A1 in both the mutated and nonmutated BoNT\/A4. This indicates that these mutations do not alter the activity of BoNT\/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.\n          <\/jats:p>","DOI":"10.1128\/aem.01795-14","type":"journal-article","created":{"date-parts":[[2014,9,20]],"date-time":"2014-09-20T04:47:04Z","timestamp":1411188424000},"page":"7415-7422","update-policy":"http:\/\/dx.doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":20,"title":["Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System"],"prefix":"10.1128","volume":"80","author":[{"given":"Marite","family":"Bradshaw","sequence":"first","affiliation":[{"name":"Department of Bacteriology, University of Wisconsin\u2014Madison, Madison, Wisconsin, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William H.","family":"Tepp","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, University of Wisconsin\u2014Madison, Madison, Wisconsin, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Regina C. M.","family":"Whitemarsh","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, University of Wisconsin\u2014Madison, Madison, Wisconsin, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sabine","family":"Pellett","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, University of Wisconsin\u2014Madison, Madison, Wisconsin, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eric A.","family":"Johnson","sequence":"additional","affiliation":[{"name":"Department of Bacteriology, University of Wisconsin\u2014Madison, Madison, Wisconsin, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/mr.56.1.80-99.1992"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro3295"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jit449"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jit450"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01180-06"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-642-33570-9_1"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.73.9.5450-5457.2005"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.toxicon.2013.05.003"},{"key":"e_1_3_3_10_2","unstructured":"US Government Printing Office. 18 March 2005. 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