Prevention of Aggregation and Autocatalysis for Sustaining Biological Activity of Recombinant BoNT/A-LC Upon Long-Term Storage

Padma      Singh; Manglesh      Kumar Singh; Vinita      Chauhan; Pallavi      Gupta; Ram      Kumar Dhaked

doi:10.2174/092986611797642689

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.

Keywords: Botulinum neurotoxin, SNAP-25, endopeptidase, autocatalysis, aggregation, glycerol, LC, HC, BoNT, rBoNT/A-LC, VAMP, MALDI-TOF, HRP, HUPOBotulinum neurotoxin, SNAP-25, endopeptidase, autocatalysis, aggregation, glycerol, LC, HC, BoNT, rBoNT/A-LC, VAMP, MALDI-TOF, HRP, HUPO