Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa, 252-8510, Japan.
Mol Genet Genomics. 2009 Dec;282(6):607-16. doi: 10.1007/s00438-009-0490-2. Epub 2009 Oct 16.
We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB (TG1) and attP (TG1) sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of phi C31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP (TG1) and attB (TG1) sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB (TG1) and attP (TG1) sites irrespective of their substrate topology. The minimal sequences of attP (TG1) and attB (TG1) sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.
我们之前已经表明，在体内，基于编码 TG1 整合酶和相应 attB（TG1）和 attP（TG1）位点的基因的整合系统不仅在链霉菌菌株中而且在大肠杆菌中都能很好地工作。此外，TG1 整合酶的附着位点与 phi C31 整合酶的附着位点不同。在本报告中，我们将 TG1 整合酶表达为 GST-TG1 整合酶融合蛋白，然后使用亲和分离和特异性切割来释放纯化的整合酶。使用纯化的 TG1 整合酶及其同源 attP（TG1）和 attB（TG1）位点建立了体外重组的条件。TG1 整合酶有效地催化 attB（TG1）和 attP（TG1）位点之间的特异性重组，而与它们的底物拓扑结构无关。证明 TG1 整合酶底物所需的 attP（TG1）和 attB（TG1）位点的最小序列分别为 43 和 39 个碱基对。这些结果为 TG1 整合酶系统提供了基本特征，可将其用作生物技术工具，以及阐明丝氨酸整合酶的机制。
