Production of the anti-HIV protein SLPI in plants

描述（由申请人提供）：一种用于人抗HIV蛋白质的新的低成本生产方法概述：人分泌性白细胞蛋白酶抑制剂（SLPI）是一种11.7kDa的粘膜蛋白质，其抗微生物（包括抗HIV）、伤口愈合和抗炎（包括抗关节炎）活性是众所周知的。SLPI蛋白天然存在于唾液和人体其他地方。具有高水平这种蛋白质的个体显著减少了艾滋病毒的传播。目前，R&D Systems，Inc.在E.大肠杆菌，并以每克约250万美元的价格出售该产品，这限制了其使用。与SLPI相关的高成本反映了高需求与有限供应的结合，考虑到在大肠杆菌中表达的非糖基化阳离子蛋白的生产。大肠杆菌需要非常广泛的变性和复性以将这种富含二硫键的蛋白质重折叠成其正常的生物活性形式。虽然在杆状病毒启动子（bv）的调控下，生物活性的人SLPI已成功地在昆虫细胞中过表达为16.4KDa的“多组氨酸标记的”蛋白（HisSLPI），但昆虫细胞不能像E.并且对于蛋白质的商业生产具有其它缺点。大约20克的SLPI将是必要的一个良好的规模临床前试验，提出了一个严峻的财政挑战，彻底调查的艾滋病毒抑制性能的SLPI。在这个STTR项目中，Edenspace Systems公司提出了一种新的、低成本的生产生物活性SLPI的方法，以促进这种有趣的人类抗HIV蛋白的临床前测试和潜在的商业生产。Edenspace的研究伙伴，密歇根州立大学的Mariam Sticklen博士，已经开发出一种植物特异性的构建体，含有SLPI基因，带有用于纯化的多组氨酸标签。该HisSLPI构建体已经整合到烟草基因组中，并且已经证明多个株系表达HisSLPI转录物。研究工作正在进行中，以确定植物特异性构建体是否产生具有生物活性的正确SLPI蛋白。在阶段I中，HisSLPI构建体将被修饰以将蛋白质靶向植物细胞中的几种不同细胞器，从而优化SLPI的产生和积累。将从烟草生物质中纯化SLPI蛋白，并检查其稳定性和抗蛋白酶的活性。在从烟草中成功地进行生物活性SLPI的第一阶段纯化之后，蛋白质的生产将按比例扩大，生产成本目标为每克1，000美元，用于第二阶段期间蛋白质抗HIV特性的临床前研究。STTR项目目标的实现有望为低成本商业化生产兆克数量的潜在重要的新型抗艾滋病毒药物奠定基础。在II期项目结束时，将向马约诊所HIV研究中心提供至少20克纯化的SLPI进行临床前试验（见随附的马约诊所Andrew Badley博士的信）。人类蛋白SLPI从唾液中分离出来，并显示出减少HIV复制和阻断HIV与膜联蛋白II（巨噬细胞表面上的受体）结合。虽然SLPI是一个有吸引力的目标，以发展为艾滋病毒治疗，它目前是不经济的，以产生足够的数量用于临床前测试。通过使用转基因烟草植物生产SLPI，Edenspace建议与研究合作伙伴一起生产足够的SLPI用于临床前试验，并开发生产系统以生产SLPI用于大规模（千克）纯化。

DESCRIPTION (provided by applicant): A New, Low-Cost Production Method for a Human Anti-HIV Protein SUMMARY: The human secretory leukocyte protease inhibitor (SLPI) is an 11.7 kDa mucosal protein well known for its anti-microbial (including anti-HIV), wound healing and anti-inflammatory (including anti- arthritis) activities. SLPI protein is found naturally in saliva and elsewhere in the human body. Individuals with high levels of this protein have notably reduced HIV transmission. At present, R&D Systems, Inc. produces this protein in E. coli and sells the product at a cost of approximately $2.5 million per gram, which restricts its use. The high cost associated with SLPI reflects high demand combined with limited supply, given that production of the non-glycosylated cationic protein expression in E. coli requires very extensive denaturation and renaturation to refold this disulfide-rich protein in its normal biologically active form. While biologically active human SLPI has been successfully overexpressed as a 16.4 KDa "polyhistidine-tagged" protein (HisSLPI) under regulation of baculovirus promoter (bv) in insect cells, insect cells cannot grow as fast as E. coli and have other drawbacks for commercial production of proteins. About 20 grams of SLPI will be necessary for a good size preclinical test, posing a severe financial challenge to thoroughly investigate of the HIV inhibitory properties of SLPI. In this STTR project, Edenspace Systems Corporation proposes to generate a new, low cost method of producing biologically active SLPI to facilitate preclinical testing and potential commercial production of this intriguing human anti-HIV protein. Edenspace's research partner, Dr. Mariam Sticklen at Michigan State University, has developed a plant-specific construct containing the gene for SLPI, with a polyhistidine tag for purification. This HisSLPI construct has been integrated into the tobacco genome, and multiple lines have been demonstrated to express the HisSLPI transcript. Work is in progress to see whether the plant-specific construct produces the correct SLPI protein that is biologically active. In Phase I, the HisSLPI construct will be modified to target the protein to several different organelles in the plant cell, thereby optimizing SLPI production and accumulation. SLPI protein will be purified from the tobacco biomass and examined for stability and activity against proteases. Following successful Phase I purification of biologically active SLPI from tobacco, production of the protein will be scaled up with a production cost objective of $1,000 per gram for preclinical studies of the anti-HIV properties of the protein during Phase II. Achievement of the goals of this STTR project is expected to provide the basis for low-cost commercial production of megagram quantities of a potentially important new anti-HIV agent. At the end of the Phase II project, at least 20 grams of purified SLPI will be provided to the Mayo Clinic HIV Research Center for preclinical testing (see attached letter from Dr. Andrew Badley of the Mayo Clinic). The human protein SLPI was isolated from saliva and shown to reduce HIV replication and block HIV binding to annexin II, a receptor on the surface of macrophages. While SLPI is an attractive target to develop as an HIV therapy, it is currently uneconomical to produce sufficient quantities for preclinical testing. By producing SLPI using transgenic tobacco plants, Edenspace proposes to manufacture enough SLPI for a preclinical trial with a research partner and develop a production system to produce SLPI for large scale (kilogram) purification.