Purification of the N- and C-terminal subdomains of recombinant heavy chain fragment C of botulinum neurotoxin serotype C.
Purification of the N- and C-terminal subdomains of recombinant heavy chain fragment C of botulinum neurotoxin serotype C.
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肉毒杆菌神经毒素血清型 C 的重组重链片段 C 的 N 端和 C 端亚结构域的纯化。
DOI：
10.1007/978-1-59745-456-8_6
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
Meagher,MichaelM
中科院分区：
文献类型：
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作者：
Huang,Jicai;Barent,Rick;Inan,Mehmet;Gouthro,Mark;Roxas,VirginiaP;Smith,LeonardA;Meagher,MichaelM
将C型肉毒毒素重链C片段（rBoNT（HC））的N-末端和C-末端部分在毕赤酵母中表达并通过离子交换层析（IEC）纯化。N-末端片段rBoNTC（Hc）-N在三个IEC步骤中纯化：Q琼脂糖凝胶快速流（FF）捕获步骤，随后是阴性SP琼脂糖凝胶FF步骤，最后，Q琼脂糖凝胶FF作为抛光步骤。基于SDS-PAGE，纯化过程产生大于90%纯的rBoNTC（Hc）-N，并且产生高达1.02g的rBoNTC（Hc）-N/kg细胞。或者，通过使用SP琼脂糖凝胶FF捕获步骤，然后是第二个SP琼脂糖凝胶FF步骤，最后是Q琼脂糖凝胶FF作为精制步骤，纯化C-末端片段rBoNTC（Hc）-C。该纯化过程产生基于SDS-PAGE的大于95%纯的rBoNTC（Hc）-C，并且产生高达0.2g的rBoNTC（Hc）-C/kg细胞。最终的蛋白质产量是发酵过程中蛋白质表达水平和纯化方法的函数，并且通常0.1和2 mg/g细胞之间的最终蛋白质产量是可接受的。另一个问题是蛋白质降解。特别是毕赤酵母，在细胞裂解和纯化过程中的蛋白酶活性一直是一个问题。N-末端降解的重要性取决于产物及其功能。N-末端测序显示纯化的rBoNTC（Hc）-N缺失蛋白质N-末端的前8个氨基酸，而纯化的rBoNTC（Hc）-C蛋白是完整的。在小鼠生物测定试验后，完整的rBoNTC（Hc）-C和缺失N-末端前8个氨基酸的rBoNTC（Hc）-N均具有疫苗效力;因此，部分降解对这些蛋白质的效用没有影响。
The N-terminal and C-terminal portions of the heavy chain fragment C from botulinum neurotoxin serotype C [rBoNT(HC)] were expressed inPichia pastorisand purified by ion-exchange chromotography (IEC). The N-terminal fragment, rBoNTC(Hc)-N, was purified in three IEC steps: a Q Sepharose Fast Flow (FF) capture step followed by a negative SP Sepharose FF step, and finally, Q Sepharose FF as a polishing step. The purification process resulted in greater than 90% pure rBoNTC(Hc)-N based on SDS-PAGE, and yielded up to 1.02 g of rBoNTC(Hc)-N/kg of cells. Alternately, the C-terminal fragment, rBoNTC(Hc)-C, was purified by using a SP Sepharose FF capture step followed by a second SP Sepharose FF step, and finally a Q Sepharose FF as a polishing step. This purification process resulted in greater than 95% pure rBoNTC(Hc)-C based on SDS-PAGE, and yielded up to 0.2 g of rBoNTC(Hc)-C/kg cells. The final protein yield is a function of protein expression level during fermentation and the purification methods, and usually final protein yield between 0.1 and 2 mg/g cells is acceptable. Another concern is protein degradation. Especially withPichia, protease activity during cell lysis and purification is always an issue. The importance of N-terminal degradation depends on product and its function. N-terminal sequencing revealed that the purified rBoNTC(Hc)-N is missing the first eight amino acids of the N-terminus of the protein, whereas the purified rBoNTC(Hc)-C protein is intact. After a mouse bioassay test, both the intact rBoNTC(Hc)-C and the rBoNTC(Hc)-N missing the first eight amino acids of the N-terminus have vaccine potency; consequently, partial degradation did not have an impact on these protein’s utility.