Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli.
Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli.
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DOI：
10.1016/j.peptides.2010.08.008
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发表时间：
2010-11
期刊：
影响因子：
3
通讯作者：
Kalman D
中科院分区：
文献类型：
--
作者：
Bommarius B;Jenssen H;Elliott M;Kindrachuk J;Pasupuleti M;Gieren H;Jaeger KE;Hancock RE;Kalman D
阳离子抗微生物宿主防御肽（HDPs）通过直接杀死多种微生物和/或调节宿主免疫来对抗感染。HDP对耐药细菌、病毒甚至寄生虫具有巨大的治疗潜力，但其治疗应用存在实质性障碍。与氨基酸前体相关的高制造成本限制了通过工业规模的化学合成递送廉价的治疗剂。相反，通过重组DNA技术在细菌中生产肽受到这些肽的抗微生物活性及其对蛋白水解降解的敏感性的阻碍，而重组肽的后续纯化通常需要多个步骤并且不具有成本效益。在这里，我们开发了适合于大规模工业生产HDP的方法;具体地，我们描述了（i）使用与SUMO的融合物在细菌中生产高产率的完整重组HDP而没有显著毒性的方法;和（ii）适合于工业用途的简化的2步纯化方法。迄今为止，我们已经使用这种方法生产了七种HDPs（IDR 1、MX 226、LL 37、CRAMP、HHC-10、E5和E6）。使用该技术，进行中试规模发酵（10 L），以生产大量的生物活性阳离子肽。总之，这些数据表明，这种新方法代表了一种具有成本效益的手段，使商业企业能够在良好实验室生产规范（GMP）条件下大规模生产用于人类治疗应用的HDPs。
Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.
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影响因子：
64.8
作者：
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