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

项目概述：我们的总体目标是开发和测试一个选择系统，用于不断发展的标签， 使细胞因子在E.大肠杆菌中表达，并容易纯化无标签的天然蛋白质。选择是 基于未折叠蛋白质限制大肠杆菌生长的发现，共表达时， 用一种工程限制性蛋白酶一种标签，其便于折叠否则折叠不好的物品， 蛋白质拯救生长。使用选择方法，我们将开发能够纯化关键的标签。 细胞因子需要创建定义，合成细胞培养基。我们的方法采用随机 蛋白G结构域的串联标签（GA-GB）的诱变和使用限制性蛋白酶的选择 我们将开发的系统。我们将为四个目标进化折叠标签，并扩展 可以在大肠杆菌中纯化的重组蛋白。具体目的是：构建共表达质粒， 用限制性蛋白酶表达GA-GB-目的蛋白;构建GA-GB-目的蛋白的随机文库; 靶蛋白;选择逃逸突变体;分析允许逃逸的突变;使用 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 这取决于我们是否能为所有四个目标进化出有效的折叠标签。一种有效的折叠标签， 定义为每L培养物可纯化≥ 50 mg天然生物活性细胞因子。在 在第一阶段结束时，我们将开发出识别两种主要的有效折叠标签的方法。 细胞因子的结构类别。我们还将了解到， 细胞因子的种类是相似的。这一知识将简化新细胞因子的折叠标签的鉴定 目标以及其他高价值蛋白质。在第二阶段，我们将为17个主要的细胞因子进化标签， 用于细胞培养。

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.