Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion.
Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion.
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在目标基因中随机位置的三重核苷酸去除：TEM-1β-内酰胺酶对氨基酸缺失的耐受性。
DOI：
10.1093/nar/gni077
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发表时间：
2005-05-16
影响因子：
14.9
通讯作者：
Jones, D Dafydd
中科院分区：
文献类型：
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作者：
Jones, D Dafydd
氨基酸的缺失是大自然适应蛋白质功能的进化机制之一。已经开发出一种简单的方法，通过在目标基因的随机位置引入恰好三个核苷酸的缺失来在体外模拟这一事件。该方法涉及对 mini-Mu 转座子进行工程改造，以引入限制性内切酶 MlyI 的识别序列。新的转座子 MuDel 能够有效插入目标 DNA 序列。为了确定该方法的功效，使用编码 TEM-1 β-内酰胺酶的 bla 基因作为靶标，并创建了一个包含 22 个不同序列变体的小型文库。在这 22 个变体中，有 8 个被鉴定出赋予大肠杆菌对氨苄西林的抗性。每种 TEM-1 变体都具有不同的氨苄青霉素最低抑制浓度，范围为 500 至 >10 000 μg/ml。序列分析显示，活跃的 TEM-1 变体不仅包含环缺失，还包含螺旋缺失，并且包含已知参与催化、抗生素耐药性和抑制剂结合的区域。这项新技术可应用于大多数基因，从而可以对蛋白质功能的缺失突变进行广泛的分析。
The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 β-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10 000 μg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.