SUMO Fusions to Enhance Expression and Secretion of Protiens

SUMO 融合增强蛋白质的表达和分泌

• 批准号: 7053070
• 负责人: Tauseef R. Butt
• 金额: $ 53.11万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053070
• 关键词: Bacillus subtilis; Escherichia coli; Streptococcus lactis; affinity chromatography; binding proteins; bioengineering /biomedical engineering; chimeric proteins; endopeptidases; functional /structural genomics; gene expression; genetic library; method development; protein purification; protein quantitation /detection; proteolysis; secretion; transfection /expression vector; ubiquitin

DESCRIPTION (provided by applicant): As more proteins are identified from the genome-sequencing projects, there is an increasing need for reliable and cost efficient means for expression and purification of proteins of academic and clinical importance. LifeSensors has developed a novel SUMO-fusion system to enhance expression, and solubility of a wide variety of proteins in E. coli, and to facilitate the downstream purification of the protein via an affinity tag attached to SUMO. An important feature of the system is the protease that cleaves the SUMO-fusion from expressed proteins. The removal of the fusion leaves no N-terminal overhang, so that any desired amino acid can be cloned at the N-terminus of the expressed protein. Several research or therapeutically important proteins either are not expressed well in bacteria or are inefficiently secreted. Recent data from the Protein Structure Initiative suggest that production of correctly folded protein is a bottleneck for success in our quest for Structural Genomics. In phase I, we proposed to exploit the chaperoning properties of SUMO to enhance expression and secretion of proteins in E. coli. Recognizing the well-known protein secretion properties of Bacillus subtilis and Lactococcus lactis we also proposed to test the role of N-terminal of SUMO in secretion of protein in these gram-positive organisms. Lactococcus lactis is well known for its secretory properties and widely used in the food industry, and yet this bacteria has not been well exploited for protein secretion in the biopharmaceutical industry. Our phase I data suggest that while E. coli is good for intracellular expression, it is not the best host for protein secretion. Lactococus lactis has emerged as the most suitable host for secretion and production of large quantities of proteins. In this phase II, LifeSensors proposes a plan to build on its success with L. lactis to test a wide variety of difficult to express protein and further validate the usefulness and role of SUMO and L. lactis as a host for protein secretion. Development of an efficient secretory system in L. lactis that allows production of correctly folded, difficult to express protein will open the bottleneck for Structural Genomics. It will also create an inexpensive platform for endotoxin free protein production that will help the biopharmaceutical industry in the post-genomic era.

描述（由申请人提供）：随着从基因组测序项目中鉴定出更多蛋白质，越来越需要可靠且具有成本效益的方法来表达和纯化具有学术和临床重要性的蛋白质。 LifeSensors 开发了一种新型 SUMO 融合系统，可增强多种蛋白质在大肠杆菌中的表达和溶解度，并通过连接到 SUMO 的亲和标签促进蛋白质的下游纯化。该系统的一个重要特征是蛋白酶可以将 SUMO 融合蛋白从表达的蛋白质中裂解出来。去除融合体不会留下 N 末端突出，因此任何所需的氨基酸都可以克隆到表达蛋白的 N 末端。一些研究或治疗上重要的蛋白质要么在细菌中表达不佳，要么分泌效率低下。来自蛋白质结构计划的最新数据表明，正确折叠蛋白质的生产是我们追求结构基因组学成功的瓶颈。在第一阶段，我们建议利用 SUMO 的陪伴特性来增强大肠杆菌中蛋白质的表达和分泌。认识到枯草芽孢杆菌和乳酸乳球菌众所周知的蛋白质分泌特性，我们还建议测试 SUMO N 末端在这些革兰氏阳性生物体蛋白质分泌中的作用。乳酸乳球菌以其分泌特性而闻名，并广泛应用于食品工业，但这种细菌尚未在生物制药行业中充分利用其分泌蛋白质。我们的第一阶段数据表明，虽然大肠杆菌有利于细胞内表达，但它并不是蛋白质分泌的最佳宿主。乳酸乳球菌已成为分泌和生产大量蛋白质的最合适宿主。在第二阶段中，LifeSensors 提出了一项计划，以乳酸乳球菌的成功为基础，测试多种难以表达的蛋白质，并进一步验证 SUMO 和乳酸乳球菌作为蛋白质分泌宿主的有用性和作用。在乳酸乳球菌中开发有效的分泌系统，允许生产正确折叠的、难以表达的蛋白质，这将打开结构基因组学的瓶颈。它还将为无内毒素蛋白质生产创建一个廉价的平台，这将有助于后基因组时代的生物制药行业。