A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease.
A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease.
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蛋白质中允许位点的简单筛选：大肠杆菌乳胶通透酶分析。
DOI：
10.1006/jmbi.1996.0881
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Bailey,J
中科院分区：
文献类型：
--
作者：
Manoil,C;Bailey,J
蛋白质可以对重大突变具有显著的耐受性。容纳大的插入而不丧失功能的位点（“允许”位点）似乎通常对应于所添加的序列不破坏整体折叠的表面区域。这些位点的鉴定可以帮助工程化具有新特性的蛋白质的功能衍生物。为了筛选允许位点，我们开发了一个简单的两步程序，用于在克隆基因中产生31个密码子插入。在第一步中，通过将转座子衍生物插入靶基因中产生β-半乳糖苷酶或碱性磷酸酶基因融合体。需要β-半乳糖苷酶或碱性磷酸酶活性来固定转座子相对于靶基因的翻译阅读框。在第二步中，在体外切除大部分转座子序列，留下框内插入。插入可以靶向细胞质或输出的蛋白质序列，并且插入的序列在多种蛋白质中充当表位。作为测试案例，在大肠杆菌乳糖通透酶基因中产生一组31个密码子插入。乳糖转运活动的突变体蛋白遵循一个简单的模式：大多数的蛋白质（10/12）与插入序列被认为是面对细胞质或周质至少部分活跃，而所有的蛋白质（9/9）与插入跨膜序列是无活性的。唯一的例外是在第三个细胞质区域插入两个无活性的蛋白质。在细胞中检测到的大多数无活性蛋白质水平降低，可能是由于蛋白水解分解。因此，这些研究说明了使用新的方法来确定允许的网站，并帮助文件的显着序列的灵活性，许多亲水性环乳糖通透酶。除了筛选允许位点，31-密码子插入诱变可能是有用的表位标记蛋白质在多个内部位置，在分析膜蛋白拓扑结构，并在解剖蛋白质的结构-功能关系。
Proteins can be remarkably tolerant of major mutational changes. Sites that accomodate large insertions without loss of function (“permissive” sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding. The identification of such sites can aid in the engineering of functional derivatives of a protein with novel properties. To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertions in cloned genes. In a first step, a β-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon derivative into the target gene. Requiring β-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the transposon relative to the target gene. In a second step, most of the transposon sequences are excised in vitro, leaving the in-frame insertion. Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety of proteins. As a test case, a set of 31-codon insertions in the Escherichia coli lac permease gene was generated. The lactose transport activities of the mutant proteins followed a simple pattern: most of the proteins (10/12) with insertions in sequences thought to face the cytoplasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive. The only exceptions were two inactive proteins with insertions in the third cytoplasmic region. Most of the inactive proteins were detected at reduced levels in cells, presumably due to proteolytic breakdown. These studies thus illustrate the use of the new method to identify permissive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease. In addition to screening for permissive sites, 31-codon insertion mutagenesis may be useful in epitope-tagging proteins at multiple internal positions, in analyzing membrane protein topology, and in dissecting structure-function relationships in proteins.
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影响因子：
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作者：
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影响因子：
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作者：
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DOI：
--
发表时间：
1988
期刊：
影响因子：
--
作者：
M. Hofnung;H. Bedouelle;J. Boulain;J. Clement;A. Charbit;P. Duplay;K. Gehring;P. Martineau;W. Saurin;S. Szmelcman
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影响因子：
2.6
作者：
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通讯作者：
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