title: Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage.
abstract: Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.
authors.affiliation: Biotechnology division, defence research and development establishment, Jhansi Road, Gwalior-474002, India.
authors.forename: Padma
authors.initials: P
authors.lastname: Singh
authors.forename: Manglesh Kumar
authors.initials: MK
authors.lastname: Singh
authors.forename: Vinita
authors.initials: V
authors.lastname: Chauhan
authors.forename: Pallavi
authors.initials: P
authors.lastname: Gupta
authors.forename: Ram Kumar
authors.initials: RK
authors.lastname: Dhaked
mesh_terms: D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D005990:Glycerol / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism; D013574:Synaptosomes
chemical_list: D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D005990:Glycerol