title: Recombinant Listeria strains producing the nontoxic L-chain of botulinum neurotoxin A in a soluble form.
abstract: Fragments of Clostridium botulinum neurotoxin A (BoNT/A) gene (botA) were expressed in Listeria monocytogenes ATCC10527 to produce the L-chain of the toxin in a soluble form. A shuttle vector pAT19 (EmR) was used to make plasmid pAT-RL containing a botA gene fragment placed under C. botulinum ntnH-gene promoter control. The plasmid also contained a C. botulinum botR/A gene, a positive transcriptional regulator of botA. The cytoplasmic fraction of the L. monocytogenes (pAT-RL) strain was found to contain up to 3 mg/L of a soluble protein of expected size and immunologically positive towards BoNT antibodies. This is the first evidence of heterologous botA gene expression producing a soluble safe derivative of botulinum neurotoxin A needed as a molecular tool for exploratory research in neurosciences as well as a basis for raising protective immunity in humans.
authors.affiliation: Laboratory of Clostridial Infections, Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia. yv@vertiev.msk.ru
authors.forename: Y V
authors.initials: YV
authors.lastname: Vertiev
authors.forename: A G
authors.initials: AG
authors.lastname: Zdanovsky
authors.forename: A B
authors.initials: AB
authors.lastname: Shevelev
authors.forename: S A
authors.initials: SA
authors.lastname: Borinskaya
authors.forename: E L
authors.initials: EL
authors.lastname: Gening
authors.forename: T
authors.initials: T
authors.lastname: Martin
authors.forename: P A
authors.initials: PA
authors.lastname: Ivanov
authors.forename: N K
authors.initials: NK
authors.lastname: Yankovsky
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003014:Clostridium botulinum / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D008089:Listeria monocytogenes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014121:Toxoids / Q000096:biosynthesis; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D000907:Antibodies, Bacterial; D004269:DNA, Bacterial; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014121:Toxoids; D019274:Botulinum Toxins, Type A