An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.
An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.
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一种产生具有改变酶性质的蛋白质的有效方法：应用于β-内酰胺酶。
DOI：
10.1073/pnas.86.23.9094
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发表时间：
1989
影响因子：
11.1
通讯作者：
Struhl,K
中科院分区：
文献类型：
--
作者：
Oliphant,AR;Struhl,K
随机序列或高度简并的寡核苷酸对于确定蛋白质和核酸中的重要功能序列都是有用的。在这种方法中，这种寡核苷酸被用来取代所需功能所需的DNA片段，功能序列通过适当的遗传或生化选择来识别。在这里，通过克隆一个混合碱基寡核苷酸来代替编码酶活性位点17个氨基酸部分的序列，产生了500,000个[纠正]改变的β -内酰胺酶蛋白。收集的大约2000种酶能够使大肠杆菌产生氨苄西林耐药性。在用不同的β -内酰胺底物进行表征后，选择其中的58个进行进一步的研究。β -内酰胺酶对不同抗生素的特异性发生改变，对自杀抑制剂克拉维酸和舒巴坦具有抗性，并且具有温度依赖性活性。定义了负责这些改变性质以及基本酶活性的氨基酸残基。这种方法应该被证明是创造具有新特性的蛋白质的有效和通用工具，特别是在蛋白质的高分辨率结构未知的情况下。
Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 500,000 [corrected] altered beta-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various beta-lactam substrates. beta-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.