Novel fluorescent protein expression vector that simplifies the solubilization of membrane proteins

摘要 简单的蛋白质突变可能会导致影响人们生活的疾病，从而产生严重的后果。蛋白 治疗药物的发展是为了靶向和取代这些蛋白质突变，本质上是为了治愈疾病。 特别的是，膜蛋白占当今药物靶标的60%以上。然而，繁琐，时间- 消耗，而且往往无效的纯化方法阻碍了膜蛋白的纯化和 下游试验。这个应用程序的长期目标是开发一种快速和简化的 针对每个特定膜蛋白的增强表达和剪裁纯化的方法；这将 极大地增加了这一重要治疗部门的商业机会。 杆状病毒表达载体系统(BEVS)通常用于生产结构蛋白。 功能研究、疫苗和治疗，但膜蛋白表达通常很低，很难 净化。ParaTechs(www.ParaTechs.com)目前销售昆虫细胞和载体，提供更高水平的 由于延迟裂解和增加感染细胞的整体健康而导致的外源基因的表达。这项建议 寻求通过开发这一创新工具来扩展我们的杆状病毒产品线，通过将 带有新型荧光融合标签的vankyrin增强型杆状病毒表达载体系统 膜蛋白的表达和增溶。目的一是设计杆状病毒转移载体 同时含有vankyrin基因和膜蛋白的病毒融合到新的荧光标签上。这个 第二个目的是评估荧光标记在选定用于增溶的洗涤剂中的稳定性 屏幕上。目的三比较膜蛋白表达的效率和效果 以及使用与重组杆状病毒相容的新的增溶屏幕进行纯化 在第一个目标中产生。 这里开发的荧光选择技术将允许增强功能蛋白质的生产，快速 能够成功增溶和轻松优化蛋白质纯化的洗涤剂的鉴定 条件。这项创新技术将克服蛋白质开发中的两大障碍 治疗学，即表达大量纯净的、经过适当处理的膜蛋白和 使膜上的活性蛋白质溶解。

ABSTRACT Simple protein mutations can have serious consequences by causing diseases that affect people's lives. Protein therapeutics are developed to target and replace those protein mutations, and essentially to heal the disease. Distinctively, membrane proteins make up over 60% of today's drug targets. However, cumbersome, time- consuming, and often ineffectual purification methods have hampered membrane protein purification and downstream experimentation. The long-term objective of this application is to develop a rapid and simplified method for enhanced expression and tailoring purification to each specific membrane protein; this would greatly enhance commercial opportunities for this important therapeutic sector. The baculovirus expression vector system (BEVS) is commonly used for production of proteins for structure- function studies, vaccines and therapeutics, but membrane proteins are often poorly expressed and difficult to purify. ParaTechs (www.ParaTechs.com) currently markets insect cells and vectors that provide heightened expression of foreign genes due to delayed lysis and increased overall health of infected cells. This proposal seeks to expand our baculovirus product line through the development of this innovative tool by combining the vankyrin-enhanced baculovirus expression vector system with a novel fluorescent fusion tag that will aid the expression and solubilization of membrane proteins. Aim one is to design baculovirus transfer vectors and viruses harboring both the vankyrin gene and membrane proteins fused to the novel fluorescent tag. The second aim will evaluate the stability of the fluorescent tag in the detergents selected for the solubilization screen. Aim three of this application will compare the efficiency and efficacy of membrane protein expression and purification using a new solubilization screen that is compatible with the recombinant baculoviruses produced in the first aim. The here developed fluorescent selection technology will allow enhanced functional protein production, rapid identification of detergents that enable successful solubilization, and easy optimization of protein purification conditions. This innovative technology will overcome two major barriers in the development of protein therapeutics, namely the expression of large quantities of pure, properly processed membrane proteins and to solubilize active proteins from membranes.