High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics.
High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics.
复制标题
高产地制备具有独特不同酶动力学的 A 型肉毒杆菌神经毒素功能活性催化转位结构域模块。
DOI：
10.1007/s10930-017-9744-8
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Singh,BalRam
中科院分区：
文献类型：
--
作者：
Dhaliwal,HarkiranpreetKaur;Thiruvanakarasu,Nagarajan;Kumar,Raj;Patel,Kruti;Ambrin,Ghuncha;Cai,Shouwei;Singh,BalRam
肉毒杆菌神经毒素(BoNTs)是已知的毒性最强的蛋白质，可由于抑制外周胆碱能突触的神经递质释放而导致松弛肌肉瘫痪。BoNT A型(BoNT/A)是一个150kDa的蛋白质，由两个主要亚基组成：轻链(LC)和重链(HC)。LC是神经毒素催化活性所必需的，而HC的C和N末端结构域分别是细胞结合和LC跨内吞体膜转运所必需的。为了更好地了解BoNT/A中毒的结构和功能方面，我们报道了高产大肠杆菌表达系统的开发(产量是文献报道的2-20倍)，用于生产具有催化活性和易位活性的重组轻链易位结构域(RLC-TD/A)模块。从失活的重组BONT/A基因(非选择性试剂)中扩增出RLC-TD/A的开放阅读框，并用pET45b(+)载体克隆表达INE。结肠癌细胞。纯化步骤依次为亲和层析、胰酶消化、阴离子交换柱层析。我们能够提纯 >； 95%的纯度、催化活性和结构良好的折叠蛋白。纯化的LC-TD/A与全长毒素和重组轻链A的酶动力学比较表明，其与底物的亲和力介于内肽酶结构域和肉毒杆菌毒素之间。对纯化蛋白在毒性和易位检测中的潜在应用进行了讨论。
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 yieldEscherichia coliexpression 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 inE. colicells. 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.