Singh Padma, Singh Manglesh Kumar, Chauhan Vinita, Gupta Pallavi, Dhaked Ram Kumar
Biotechnology division, defence research and development establishment, Jhansi Road, Gwalior-474002, India.
Protein Pept Lett. 2011 Dec;18(12):1177-87. 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.
在蛋白质表达、纯化、储存或转移至所需分析缓冲液的过程中，蛋白质聚集会妨碍其在体外研究中的应用。这些聚集体的形成是生物活性研究中的主要障碍，也限制了可用于生物医学应用的蛋白质产品范围。A型肉毒杆菌神经毒素的催化轻链在长期储存和冻融纯化后会发生自催化和聚集。在本研究中，从合成基因优化了A型肉毒杆菌神经毒素生物活性轻链蛋白的高水平表达和纯化条件。筛选了几种共溶剂以防止重组肉毒杆菌神经毒素A轻链（rBoNT/A-LC）的自催化和聚集。研究了共溶剂对重组蛋白长期储存期间内肽酶活性的影响。还评估了纯化的rBoNT/A-LC的免疫原性。
