Production of the anti-HIV protein SLPI in plants

摘要：人分泌性白细胞蛋白酶抑制剂（SLPI）是一种11.7 kDa的粘膜蛋白，以其抗微生物（包括抗hiv）、伤口愈合和抗炎（包括抗关节炎）活性而闻名。SLPI蛋白天然存在于唾液和人体其他部位。这种蛋白水平高的个体显著降低了艾滋病毒的传播。目前，研发系统公司在大肠杆菌中生产这种蛋白质，并以每克约250万美元的价格销售产品，这限制了它的使用。与SLPI相关的高成本反映了高需求和有限的供应，因为在大肠杆菌中生产非糖基化阳离子蛋白表达需要非常广泛的变性和再变性才能将这种富含二硫化物的蛋白以其正常的生物活性形式重新折叠。虽然在杆状病毒启动子（bv）的调控下，具有生物活性的人类SLPI已成功地在昆虫细胞中以16.4 KDa的“多组氨酸标记”蛋白（HisSLPI）过表达，但昆虫细胞不能像大肠杆菌那样快速生长，并且在蛋白质的商业化生产方面存在其他缺点。大约20克的SLPI将需要一个良好的规模的临床前试验，这对彻底研究SLPI的HIV抑制特性提出了严峻的经济挑战。在这个STTR项目中，Edenspace系统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结构，其中包含SLPI基因，并带有用于纯化的多组氨酸标签。该HisSLPI构建物已被整合到烟草基因组中，并且已证明多个品系表达HisSLPI转录本。目前正在研究这种植物特异性结构是否能产生具有生物活性的正确的SLPI蛋白。在第一阶段，将对HisSLPI结构进行修饰，使其靶向植物细胞中的几种不同细胞器，从而优化SLPI的生产和积累。将从烟草生物质中纯化SLPI蛋白，并检测其稳定性和抗蛋白酶活性。在成功地从烟草中纯化出具有生物活性的SLPI后，该蛋白的生产规模将扩大，生产成本目标为每克1000美元，用于二期蛋白质抗hiv特性的临床前研究。该STTR项目目标的实现预计将为一种可能重要的新型抗艾滋病毒药物的百万克量的低成本商业生产提供基础。在二期项目结束时，至少20克纯化SLPI将提供给梅奥诊所HIV研究中心进行临床前测试（见附件梅奥诊所Andrew Badley博士的信）。从唾液中分离出的人类蛋白SLPI可减少HIV复制并阻断HIV与巨噬细胞表面受体膜联蛋白II的结合。虽然SLPI是一种有吸引力的HIV治疗靶点，但目前生产足够数量的SLPI用于临床前测试是不经济的。通过使用转基因烟草植物生产SLPI， Edenspace建议与研究伙伴一起生产足够的SLPI用于临床前试验，并开发一种大规模（公斤）纯化SLPI的生产系统。

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.