Selection system for identifying protein-specific folding tags that enable purification of native cytokines from E. coli

用于识别蛋白质特异性折叠标签的选择系统，从而能够从大肠杆菌中纯化天然细胞因子

• 批准号: 10256900
• 负责人: PHILIP N BRYAN
• 金额: $ 22.48万
• 依托单位: POTOMAC AFFINITY PROTEINS, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10256900
• 关键词: Affinity; Amino Acids; Binding; Biological; Cell Culture Techniques; Cells; Chimeric Proteins; Culture Media; DNA cassette; Data; Disulfides; Engineering; Epidermal Growth Factor; Escape Mutant; Escherichia coli; G-substrate; Genes; Goals; Growth; IL2 gene; In Vitro; Interleukins; Knowledge; LIF gene; Left; Libraries; Methods; Mutagenesis; Mutation; Peptide Hydrolases; Phase; Plasmids; Process; Production; Property; Proteins; Recombinant Proteins; Recombinants; Reproducibility; Specificity; Structure; Surface; System; Testing; Therapeutic; Transforming Growth Factors; VEGFA gene; base; cell transformation; cost; cytokine; disulfide bond; protein folding

Project Summary: Our overall objective is to develop and test a selection system for evolving tags that enable folding of cytokines in E. coli and facile purification of the tag-free, native protein. Selection is based on the discovery that unfolded proteins restrict growth of the E. coli host cell when co-expressed with an engineered restriction protease. A tag that facilitates the folding of an otherwise poorly-folded protein rescues growth. Using the selection method, we will develop tags enabling purification of key cytokines needed to create defined, synthetic cell culture media. Our approach employs random mutagenesis of a tandem tag of Protein G domains (GA-GB) and selection using a restriction protease system that we will develop. We will evolve folding tags for four targets and expand the envelop of recombinant proteins that can be purified in E coli. Specific Aims are: Construct plasmids for co- expression of GA-GB-target proteins with the restriction protease; Construct random libraries of GA-GB- target protein; Select for escape mutants; Analyze of mutations that allow escape; Purify proteins using newly-evolved tags; Test physical and biological properties of purified proteins. Feasibility will be determined by whether we can evolve effective folding tags for all four targets. An effective folding tag is defined as one that allows purification of ≥50mg of native, biologically-active cytokine per L of culture. At the end of Phase I we will have developed methods for identifying effective folding tags for two major structural classes of cytokines. We will also have learned whether evolved folding tags for a structural class of cytokine are similar. This knowledge will simplify identification of folding tags for new cytokine targets as well as other high-value proteins. In Phase II we would evolve tags for 17 major cytokines used in cell culture.

项目摘要：我们的总体目标是开发和测试用于进化标签的选择系统 使大肠杆菌中的细胞因子折叠和无标签的天然蛋白质均匀纯化 基于以下发现，即共表达时展开的蛋白质限制了大肠杆菌宿主细胞的生长 带有生成的限制蛋白酶。 蛋白质的生长使用选择方法，我们将开发标签 细胞因子需要创建定义的合成培养基。 使用限制性蛋白酶的蛋白质G结构域（GA-GB）的串联标签的诱变 我们将开发的系统。 在E大肠杆菌中纯化的重组蛋白。 具有约束蛋白的GA-GB靶向蛋白的表达； 靶蛋白；用于逃脱的突变； 新进化的标签； 通过我们是否可以为所有四个目标进化的折叠标签确定。 定义为允许≥50mg的天然，生物活性细胞因子每L培养物的纯化 第一阶段结束，我们将开发用于识别两个主要折叠标签的方法 细胞因子的结构类别。 细胞因子类相似。 靶标和其他高价值蛋白。 用于细胞培养。