Genetic Engineering of Taqi Restriction Endonuclease

该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域（结构域），它们在三维阵列中相互作用以提供生物活性。在蛋白质中插入两个氨基酸会引起在三维结构内具有一定影响半径的小扰动。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域，哪些区域充当连接这些域的铰链。这一建议提出了一种通过在编码TagI限制性内切酶的DNA序列水平上操作来构建这种插入蛋白的一般方法。目的是了解DNA序列识别特异性的TagI核酸内切酶，并工程突变与改变序列识别。野生型内切酶，即所谓的“变构激活突变体”和“规范位点突变体”，将被过量生产，纯化，并在体外鉴定序列特异性结合，解离，促进扩散和切割，使用各种DNA底物。使用一个独特的TagI核酸内切酶克隆，将额外的双密码子插入突变引入酶中，并通过体外和体外测定进行表征。这些研究将有助于确定序列特异性结构域，并允许这些结构域的进一步靶向饱和诱变。这些突变蛋白的新识别序列将使用唯一标记的DNA底物来确定。与John Anderson博士合作，纯化的TagI将与其同源寡核苷酸共结晶，以确定其三维结构。

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.