Genetic Engineering of Taqi Restriction Endonuclease

Taqi限制性内切酶基因工程

• 批准号: 8714352
• 负责人: Francis Barany
• 金额: $ 10万
• 依托单位: Joan and Sanford I. Weill Medical College of Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1990-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8714352&HistoricalAwards=false
• 关键词: Genetic; Engineering; Taqi; Restriction; Endonuclease

This project will employ a new approach to understanding protein function and design. Protein secondary structure elements fold into precise functional regions (domains), which interact in a three dimensional array to provide biological activity. Insertion of two amino acids into a protein causes a small perturbation which has a certain radius of influence within the three dimensional structure. By introducing two-amino acid insertions into the linear sequence, one can scan the length of a protein and determine which regions might comprise functional domains and which regions act as the hinges that connect the domains. This proposal presents a general method for constructing such insertions in proteins by operating at the level of the DNA sequence that codes for the TagI restriction endonuclease. The aim is to understand the specificity of DNA sequence recognition by TagI endonuclease, and to engineer mutants with altered sequence recognition. Wild type endonuclease, a so called "allosteric activation mutant", and a "canonical site nicking mutant" will be overproduced, purified, and characterized in vitro with respect to sequence specific binding, dissociation, facilitated diffusion, and cleavage, using a variety of DNA substrates. Using a unique TagI endonuclease clone, additional two-codon insertion mutations will be introduced into the enzyme, and characterized by both in vitro and in vitro assays. These studies will help to define the sequence specificity domains, and allow for further targeted saturation mutagenesis of those domains. The new recognition sequence of these mutant proteins will be determined using a uniquely labeled DNA substrate. In collaboration with Dr. John Anderson, purified TagI will be co-crystalized with its cognate oligonucleotide for determination of its three dimensional structure.

这个项目将采用一种新的方法来理解蛋白质的功能和设计。蛋白质二级结构元素折叠成精确的功能区(结构域)，这些功能区以三维阵列的形式相互作用，提供生物活性。在蛋白质中插入两个氨基酸会引起微小的扰动，在三维结构中具有一定的影响半径。通过将两个氨基酸插入到线性序列中，人们可以扫描蛋白质的长度，并确定哪些区域可能包含功能结构域，哪些区域充当连接这些结构域的铰链。这项建议提出了一种通用的方法，通过在编码Tagi限制性内切酶的DNA序列水平上操作来构建蛋白质中的这种插入。目的是了解Tagi内切酶识别DNA序列的特异性，并设计具有改变序列识别能力的突变体。野生型核酸内切酶，一个被称为“变构激活突变体”，和一个“典型部位划痕突变体”，将被大量生产、纯化，并在体外利用各种DNA底物在序列特异性结合、解离、促进扩散和切割方面进行鉴定。利用一个独特的Tagi内切酶克隆，额外的两个密码子插入突变将被引入到该酶中，并通过体外和体外检测进行鉴定。这些研究将有助于确定序列特异性结构域，并允许对这些结构域进行进一步的定向饱和突变。这些突变蛋白质的新识别序列将使用唯一标记的DNA底物来确定。在约翰·安德森博士的合作下，纯化的TAGI将与其同源寡核苷酸共结晶，以确定其三维结构。