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首页> 美国卫生研究院文献>Nucleic Acids Research >A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach
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A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

机译：一种基因截短策略可产生蛋白质的N和C端缺失变体以进行功能研究：通过基于Mu体外转座的方法对酵母Mso1p中的Sec1p结合域进行定位

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摘要

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.
机译：噬菌体Mu体外转座构成了分子生物学中的一种通用工具，其应用范围从单个基因或蛋白质的工程改造到基因组片段或整个基因组的修饰。在Mu转座的基础上设计了一种新的策略，该策略通过几个操作步骤同时生成了一组嵌套的基因构建体，它们编码蛋白质的缺失变体。使用微型Mu转座子产生C端缺失，该转座子在每个转座子末端附近携带翻译终止信号。类似地，使用具有适当限制位点的转座子产生N-末端缺失，其允许缺失基因的5'-远端部分。作为原理的证明，我们生产了一组编码酵母Mso1p的C和N端截短变体的质粒构建体，并绘制了其Sec1p相互作用区域。 Mso1p中最重要的相互作用氨基酸位于残基T46和N78之间，相互作用可能较弱，可能在E79–N105区域内。这种通用的基因截断策略非常有效，并且可以在单个反应序列中生成编码蛋白质缺失变体的基因构建的全面库，这在许多类型的功能研究中都很有价值。重要的是，该方法适用于克隆在适当载体中的任何蛋白质编码基因。

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