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Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing\/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg\/ml and purified by one-step Ni<jats:sup>+<\/jats:sup>affinity chromatography. Mice immunized with three injections containing 5 \u03bcg of any of the<jats:italic>in vitro<\/jats:italic>-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10<jats:sup>5<\/jats:sup>against the native toxin complex, which enabled protection against a high-dose toxin challenge (10<jats:sup>3<\/jats:sup>to 10<jats:sup>6<\/jats:sup>mouse 50% lethal dose [MsLD<jats:sub>50<\/jats:sub>]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10<jats:sup>5<\/jats:sup>MsLD<jats:sub>50<\/jats:sub>toxin challenge. Our results together with the latest developments in scalability of the<jats:italic>in vitro<\/jats:italic>protein expression systems offer alternative routes for the preparation of botulinum vaccine.<\/jats:p>","DOI":"10.1128\/cvi.00496-09","type":"journal-article","created":{"date-parts":[[2010,4,1]],"date-time":"2010-04-01T02:43:51Z","timestamp":1270089831000},"page":"784-792","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":57,"title":["Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System"],"prefix":"10.1128","volume":"17","author":[{"given":"R.","family":"Zichel","sequence":"first","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Mimran","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Keren","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Barnea","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"I.","family":"Steinberger-Levy","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D.","family":"Marcus","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Turgeman","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.","family":"Reuveny","sequence":"additional","affiliation":[{"name":"Department of Biotechnology"},{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.B. 19, Ness Ziona, 76100 Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.71.3.1599-1603.2003"},{"key":"e_1_3_2_3_2","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","volume":"285","year":"2001","unstructured":"Arnon, S. 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