Production of the anti-HIV protein SLPI in plants

植物中抗 HIV 蛋白 SLPI 的生产

• 批准号: 7328198
• 负责人: DAVID A LEE
• 金额: $ 9.57万
• 依托单位: EDENSPACE SYSTEMS CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7328198
• 关键词: ANXA2 gene; Achievement; Affinity Chromatography; Anti-HIV Agents; Anti-Inflammatory Agents; Anti-inflammatory; Arthritis; Baculoviruses; Binding; Biomass; Cells; Clinic; Clinical; Costs and Benefits; Disulfides; Endopeptidases; Escherichia coli; Foundations; Genes; Genome; Goals; HIV; HIV therapy; Human; Human body; In Vitro; Individual; Insecta; Kilogram; Left; Letters; Methods; Michigan; Nickel; Organelles; Peptide Hydrolases; Phase; Phase I Clinical Trials; Plant Leaves; Plants; Preclinical Testing; Process; Production; Property; Protein Overexpression; Proteins; Recombinants; Regulation; Research; Saliva; Serine Protease; Small Business Technology Transfer Research; Surface; System; Testing; Tobacco; Transcript; Transgenic Organisms; Universities; Western Blotting; Work; Wound Healing; antileukoprotease; antimicrobial; base; cost; human SLPI protein; immunocytochemistry; macrophage; polyhistidine; preclinical study; promoter; protein expression; receptor; research and development; scale up; size; transmission process

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.

描述（由申请人提供）：一种新的、低成本的人类抗 HIV 蛋白生产方法 摘要：人类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）而闻名。 HIV）、伤口愈合和抗炎（包括抗关节炎）活性。 SLPI 蛋白天然存在于唾液和人体其他部位。这种蛋白质含量高的人显着减少了艾滋病毒的传播。目前，R&D Systems, Inc. 在大肠杆菌中生产这种蛋白质，并以每克约 250 万美元的价格销售该产品，这限制了其使用。与 SLPI 相关的高成本反映了高需求和有限的供应，因为在大肠杆菌中表达非糖基化阳离子蛋白需要非常广泛的变性和复性，以将这种富含二硫键的蛋白重新折叠为其正常的生物活性形式。虽然具有生物活性的人类 SLPI 已在昆虫细胞中在杆状病毒启动子 (bv) 的调控下成功过表达为 16.4 KDa“多组氨酸标签”蛋白 (HisSLPI)，但昆虫细胞的生长速度不如大肠杆菌，并且在商业化方面还有其他缺点蛋白质的生产。进行良好尺寸的临床前测试需要约 20 克 SLPI，这对彻底研究 SLPI 的 HIV 抑制特性构成了严峻的财务挑战。在这个 STTR 项目中，Edenspace Systems Corporation 提议开发一种新的低成本方法来生产具有生物活性的 SLPI，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 的研究合作伙伴、密歇根州立大学的 Mariam Sticklen 博士开发了一种包含 SLPI 基因的植物特异性构建体，并带有用于纯化的多组氨酸标签。该 HisSLPI 构建体已整合到烟草基因组中，并且多个品系已被证明可以表达 HisSLPI 转录本。目前正在研究植物特异性构建体是否产生具有生物活性的正确 SLPI 蛋白。在第一阶段，HisSLPI 构建体将被修改，以将蛋白质靶向植物细胞中的几个不同的细胞器，从而优化 SLPI 的生产和积累。 SLPI 蛋白将从烟草生物质中纯化，并检查其稳定性和抗蛋白酶活性。在成功完成第一阶段从烟草中纯化生物活性 SLPI 后，该蛋白质的生产将扩大，生产成本目标为每克 1,000 美元，用于第二阶段蛋白质抗 HIV 特性的临床前研究。该 STTR 项目目标的实现预计将为兆克数量的低成本商业生产潜在重要的新型抗艾滋病毒药物奠定基础。在二期项目结束时，至少20克纯化的SLPI将提供给梅奥诊所HIV研究中心用于临床前测试（见附上梅奥诊所Andrew Badley博士的信件）。从唾液中分离出人类蛋白质 SLPI，显示其可以减少 HIV 复制并阻止 HIV 与膜联蛋白 II（巨噬细胞表面的受体）结合。虽然 SLPI 是作为 HIV 疗法开发的一个有吸引力的靶点，但目前生产足够数量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 计划与研究合作伙伴一起生产足够的 SLPI 用于临床前试验，并开发生产系统来生产用于大规模（公斤）纯化的 SLPI。