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.