Dhaliwal Harkiranpreet Kaur, Thiruvanakarasu Nagarajan, Kumar Raj, Patel Kruti, Ambrin Ghuncha, Cai Shouwei, Singh Bal Ram
Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, North Dartmouth, MA, 02747, USA.
Department of Pharmaceutical Sciences, Northeastern University, Boston, 02115, USA.
Protein J. 2017 Dec;36(6):489-501. doi: 10.1007/s10930-017-9744-8.
Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2-20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify > 95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.
肉毒杆菌神经毒素（BoNTs）是已知毒性最强的蛋白质，可通过抑制外周胆碱能突触释放神经递质导致弛缓性肌肉麻痹。A型肉毒杆菌神经毒素（BoNT/A）是一种150 kDa的蛋白质，由两个主要亚基组成：轻链（LC）和重链（HC）。神经毒素的催化活性需要轻链，而重链的C端和N端结构域分别是细胞结合以及轻链跨内体膜转运所必需的。为了更好地理解BoNT/A中毒的结构和功能方面，我们在此报告了一种高产大肠杆菌表达系统的开发（产量比文献报道的值高2至20倍），用于生产具有催化活性和转运能力的BoNT/A重组轻链 - 转运结构域（rLC-TD/A）模块。rLC-TD/A的开放阅读框从失活的重组BoNT/A基因（一种非选择性试剂）中通过PCR扩增得到，并使用pET45b(+)载体进行克隆，以便在大肠杆菌细胞中表达。纯化过程包括亲和色谱、胰蛋白酶消化和阴离子交换柱色谱的顺序步骤。我们能够纯化出纯度大于95%、具有催化活性且结构良好折叠的蛋白质。将纯化的LC-TD/A与全长毒素和重组轻链A的酶动力学进行比较表明，其对底物的亲和力介于内肽酶结构域和肉毒杆菌毒素之间。已在毒性和转运测定中讨论了纯化蛋白质的潜在应用。
