Dubin Manu J, Bowler Chris, Benvenuto Giovanna
Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy.
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.prot5401. doi: 10.1101/pdb.prot5401.
In recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.
近年来，诸如Gateway系统之类的序列特异性重组克隆方法在包括植物在内的许多不同生物体的高通量研究中用于（过）表达带标签的蛋白质时越来越受欢迎。由于其通用性和易用性，这些方法在低通量和中通量研究中也受到青睐。然而，由于重组步骤，所产生的融合蛋白含有长的且通常带高电荷的多克隆位点序列，这些序列可能会干扰其生理功能。此外，在某些情况下，为了进行N端和C端标记，感兴趣的基因必须克隆两次（一次带有终止密码子，一次不带终止密码子）。在此，我们提出了一种混合组合克隆策略，该策略克服了许多这些限制。在第一步中，将感兴趣的基因克隆到一个含有标准化克隆位点的入门载体中，这些克隆位点带有所需的N端或C端标签以及优化的多克隆位点序列。使用Gateway重组反应将蛋白质标签融合体从入门克隆转移到具有所需启动子和针对感兴趣生物体的选择标记的Gateway目的载体中。随着实验要求的不断发展，可以快速轻松地构建用于表达具有所需标签、启动子、选择标记或其他特征的感兴趣蛋白质的构建体。
