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不断发展的标签的选择系统 使大肠杆菌中的细胞因子折叠和无标签的天然蛋白质的便捷纯化。选择是 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 具有工程限制蛋白酶。一个有助于折叠不佳的折叠的标签 蛋白质反应生长。使用选择方法，我们将开发标签，以纯化密钥 细胞因子需要创建定义的合成细胞培养基。我们的方法员工随机 使用约束蛋白酶的蛋白质G结构蛋白（GA-GB）串联标签的诱变 我们将开发的系统。我们将进化四个目标的折叠标签，并扩展 重组蛋白可以在E大肠杆菌中纯化。具体目的是：构造共同的质粒 具有限制蛋白的GA-GB靶蛋白的表达；构建GA-GB-的随机库 靶蛋白；选择逃生突变体；分析允许逃脱的突变；使用纯化蛋白质 新进化的标签；测试纯化蛋白质的物理和生物学特性。可行性将是 确定我们是否可以为所有四个目标进化有效的折叠标签。有效的折叠标签是 定义为允许每L培养物纯化≥50mg的天然，生物活性细胞因子。 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。我们还将了解结构上是否进化的折叠标签 细胞因子类别相似。这些知识将简化新细胞因子的折叠标签的识别 靶标以及其他高价值蛋白。在第二阶段，我们将进化17个主要细胞因子的标签 用于细胞培养。