Directed Protein Evolution for Design of LAGLIDADGs with Novel Recognition Speci

脊椎动物免疫系统利用的突变和选择的迭代循环是一个强大的 用于产生具有不同性质的蛋白质的方法。为了将这种方法应用于生成 LAGLIDADG归巢核酸内切酶（LHE）具有新的DNA结合和切割特异性，我们 开发了在培养的B细胞表面上将LHE表达为融合蛋白的方法。表面 表达的融合蛋白允许快速评估特异性结合和切割特性， 也可用于分离表达LHE的细胞群 有不同的特异性根据这些数据，我们提出了以下具体目标：在具体目标1中， 我们将根据新的LHE生成和表征新的表面表达LHE支架， 组件2（Monnat）和组件3（Baker）。在具体目标2中，我们将整合表面 将LHE表达到DT 40细胞系的免疫球蛋白基因座中，使得它们变得对 在DT 40细胞中操作的内源性体细胞超突变机制。然后我们将利用这些LHE超变 行来执行两种迭代突变/选择策略，以鉴定新的LHE， 所需的结合和切割性质。首先，我们将使用来自组件3（Baker）的LHE， 预优化的DNA/蛋白质界面朝向靶位点，并将尝试直接选择LHE变体 能够结合和切割预测的靶。在第二阶段，我们将尝试一个碱基对一个碱基对的迁移， 策略，从目前可用的表面表达的LHE支架开始。在具体目标3中，我们 进一步完善和加强我们的方法和技术，用于迭代突变/选择， LHE变体。在此目标的一部分中，我们将使用组件2（Monnat）和组件5 （Stoddard）开发了一种基于量子点纳米传感器的灵敏度提高的切割测定法， 用于流式细胞术和可溶性LHE测定。第二种，我们将利用过表达 参与体细胞超突变的蛋白质，以增加超突变的速率并增强谱 包括更高的插入和缺失率。 这一目标的输出也直接反馈到NGEC LHE设计周期，作为识别的变体 这里将被传递到组件2（Monnat）和组件5（Stoddard）进行生化和 生物物理分析，并将由此获得的信息纳入PSSM矩阵，以确定最佳 工程网站的组件2（Monnat），并进入计算设计算法开发的 组成部分3（面包师）。

The iterative cycling of mutation and selection utilized by vertebrate immune systems is a powerful means for generating proteins with diverse properties. In order to apply this approach to the generation of LAGLIDADG homing endonucleases (LHEs) with novel DNA binding and cleavage specificities, we have developed methods to express LHEs as fusion proteins on the surface of cultured B-cells. The surface expressed fusion proteins allow the rapid assessment of the specific binding and cleavage properties of the LHE using flow cytometry, and also can be used to separate populations of cells expressing LHE's with different specificities. Based on these data, we propose the following Specific Aims: In Specific Aim 1, we will generate and characterize new surface expressed LHE scaffolds based on novel LHE's generated by Component 2 (Monnat) and Component 3 (Baker). In Specific Aim 2, we will integrate surface expressed LHE's into immunoglobulin loci of the DT40 cell line such that they become susceptible to the endogenous somatic hypermutation mechanism(s) operating in DT40 cells. We will then use these LHEhypermutating lines to execute two strategies of iterative mutation/selection to identify novel LHE's with desired binding and cleavage properties. In the first, we will use LHE's from Component 3 (Baker) with pre-optimized DNA/protein interfaces towards target sites, and will attempt to directly select LHE variants able to bind and cleave the predicted target. In the 2nd, we will attempt a base pair-by-base pair migration strategy, beginning with presently available surface expressed LHE scaffolds. In Specific Aim 3, we will work on further refining and enhancing our methods and technologies for iterative mutation/selection of LHE variants. In one part of this aim, we will work with Component 2 (Monnat) and Component 5 (Stoddard) on developing an increased sensitivity cleavage assay based on quantum dot nanosensors, for use in both flow cytometry and soluble LHE assays. In the second, we will utilize overexpression of proteins involved in somatic hypermutation to incrase the rate of hypermutation and enhance the spectrum of mutations to include a higher rate of insertions and deletions. The output of this aim directly feeds back to the NGEC LHE design cycle as well, as variants identified here will be passed on to Component 2 (Monnat) and Component 5 (Stoddard) for biochemical and biophysical analysis, with information thus derived incorporated into PSSM matrices for identifying the best engineerable sites by Component 2 (Monnat), and into computational design algorithms developed by Component 3 (Baker).