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

新型荧光蛋白表达载体，可简化膜蛋白的溶解

基本信息

批准号：
9462412
负责人：
Angelika Fath-Goodin
金额：
$ 22.5万
依托单位：
PARATECHS CORP.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-20 至 2019-03-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9462412
关键词：
Affect Antigens Baculoviridae Baculoviruses Biological Assay Cell Communication Cells Charge Chimeric Proteins Cloning Column Chromatography Complex Cytolysis Detergents Development Disease Drug Targeting Elements Failure Gene Proteins Genes Health Incubated Infection Insecta Life Lipid Bilayers Literature Mammalian Cell Membrane Proteins Methods Mutation Nickel Outcome Phase Process Production Property Proteins Reagent Recombinants Research Sampling Signal Transduction Solubility Structure System Technology Testing Therapeutic Time Transport Process Ultraviolet Rays Vaccines Virus Western Blotting base commercial application cost effective design expression vector healing improved innovation innovative technologies interest laboratory equipment novel prevent protein aggregate protein expression protein function protein purification protein structure function screening stability testing synergism technological innovation therapeutic protein tool vector

项目摘要

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.

抽象的简单的蛋白质突变可能会导致影响人们生活的疾病，从而产生严重后果。蛋白质开发治疗方法是为了针对和替代这些蛋白质突变，本质上是为了治愈疾病。值得注意的是，膜蛋白占当今药物靶标的 60% 以上。不过比较麻烦，费时间消耗且通常无效的纯化方法阻碍了膜蛋白的纯化和下游实验。该应用程序的长期目标是开发一种快速且简化的针对每种特定膜蛋白增强表达和定制纯化的方法；这会极大地增加了这一重要治疗领域的商业机会。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、疫苗和治疗方法，但膜蛋白往往表达不良且难以净化。 ParaTechs (www.ParaTechs.com) 目前销售昆虫细胞和载体，可提供增强的由于延迟裂解和受感染细胞整体健康状况的提高而导致外源基因的表达。这个提议旨在通过开发这种创新工具来扩大我们的杆状病毒产品线，结合 vankyrin 增强的杆状病毒表达载体系统，带有新型荧光融合标签，有助于膜蛋白的表达和溶解。目标一是设计杆状病毒转移载体并病毒携带有与新型荧光标签融合的vankyrin基因和膜蛋白。这第二个目标是评估荧光标签在所选用于增溶的洗涤剂中的稳定性屏幕。该应用的第三个目标将比较膜蛋白表达的效率和功效并使用与重组杆状病毒兼容的新溶解筛选进行纯化产生于第一个目标。这里开发的荧光选择技术将增强功能性蛋白质的生产，快速鉴定能够成功溶解并轻松优化蛋白质纯化的去污剂状况。这项创新技术将克服蛋白质开发的两大障碍疗法，即表达大量纯的、经过适当加工的膜蛋白并溶解膜上的活性蛋白质。