{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T23:50:34Z","timestamp":1790639434566,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Areas Research and Development Program of Guangdong Province","award":["2024B1111160006"],"award-info":[{"award-number":["2024B1111160006"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxins (BoNTs) act on peripheral cholinergic nerve terminals, inducing reversible muscle paralysis and profound therapeutic effects. However, their limited cell-type specificity and narrow therapeutic window have motivated the development of engineered variants. Here, a modular strategy was employed to construct full-length chimeric BoNTs, grafting receptor-binding segments from BoNT\/B or BoNT\/F onto the BoNT\/A framework. The novel chimeras AAAF and AAFF efficiently cleaved rSNAP-25 in cell-free assays. Firstly, both toxins showed effective cellular uptake and cleaved endogenous SNAP-25 in Neuro-2a cells, with cleavage efficiencies of approximately 46% for AAAF and 73% for AAFF, highlighting the enhanced activity of AAFF. Secondly, AAAF induced faster recovery from reversible muscle paralysis compared to rBoNT\/A-WT, whereas AAFF produced more sustained paralysis, with both exhibiting reduced systemic toxicity. Despite these altered pharmacological profiles, the chimeras required higher doses than rBoNT\/A-WT to induce neuromuscular effects. Collectively, this study presents the design of novel chimeric BoNT\/A-F proteins, characterizes their functional activities, and provides a preliminary exploration of how domain grafting affects cellular uptake, enzymatic activity, and neuromuscular pharmacodynamics.<\/jats:p>","DOI":"10.3390\/toxins18050205","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T09:46:11Z","timestamp":1777455971000},"page":"205","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Design and Characterization of Recombinant and Chimeric BoNT\/A Neurotoxins with Receptor-Binding Domain Grafting"],"prefix":"10.3390","volume":"18","author":[{"given":"Sihan","family":"Pan","sequence":"first","affiliation":[{"name":"School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanzhi","family":"Ye","sequence":"additional","affiliation":[{"name":"School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Li","sequence":"additional","affiliation":[{"name":"School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongxin","family":"Fu","sequence":"additional","affiliation":[{"name":"School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China"},{"name":"Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9908-1001","authenticated-orcid":false,"given":"Jufang","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China"},{"name":"Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1124\/pr.116.012658","article-title":"Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology","volume":"69","author":"Pirazzini","year":"2017","journal-title":"Pharmacol. 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