Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage

• Published in: Protein and peptide letters Vol. 18; no. 12; p. 1177
• Main Authors: Singh, Padma, Singh, Manglesh Kumar, Chauhan, Vinita, Gupta, Pallavi, Dhaked, Ram Kumar
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2011
• Subjects: Blotting, Western; Botulinum Toxins, Type A - chemistry; Botulinum Toxins, Type A - metabolism; Catalysis; Endopeptidases - chemistry; Endopeptidases - metabolism; Escherichia coli; Glycerol - pharmacology; Protein Denaturation - drug effects; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Synaptosomal-Associated Protein 25 - chemistry; Synaptosomal-Associated Protein 25 - metabolism; Synaptosomes
• ISSN: 1875-5305
• DOI: 10.2174/092986611797642689

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.