Xu Shujing, Ju Jiansong, Misono Haruo, Ohnishi Kouhei
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan.
Gene. 2006 Mar 1;368:126-37. doi: 10.1016/j.gene.2005.10.032. Epub 2006 Jan 23.
RecA-dependent homologous recombination in Escherichia coli is a very effective way to construct chimeras between two homologous genes. The disadvantage of in vivo method is a small library size of chimeric genes in comparison with in vitro DNA shuffling. In order to overcome the disadvantage, we have developed novel in vivo DNA shuffling methods with successive homologous recombinations. Linearized DNA molecules with two homologous genes were made with ligation rather than the conventional restriction enzyme cleavage between two genes. The three-way ligation of a vector and two homologous bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases or the two-way ligation of the donor bphC gene and an acceptor plasmid carrying the homologous bphC gene generated a variety of linearized DNA molecules. The homologous recombination between the genes on the linearized DNA molecules created the large chimeric bphC gene libraries in a recBC sbcA E. coli strain. After three rounds of recombinations, chimeric bphC genes with four-part gene fragments by triple-crossover were easily obtained. By employing a 96-well microtiter plate high-throughput screening, thermally stable chimeric 2,3-dihydroxybiphenyl 1,2-dioxygenases were selected from chimeric bphC gene libraries. This opens up a new way for directed evolution of proteins in vivo.
大肠杆菌中依赖RecA的同源重组是构建两个同源基因之间嵌合体的一种非常有效的方法。与体外DNA改组相比，体内方法的缺点是嵌合基因文库规模较小。为了克服这一缺点，我们开发了具有连续同源重组的新型体内DNA改组方法。具有两个同源基因的线性化DNA分子通过连接而非传统的基因间限制性酶切制备。载体与两个编码2,3-二羟基联苯1,2-双加氧酶的同源bphC基因的三向连接，或供体bphC基因与携带同源bphC基因的受体质粒的双向连接，产生了多种线性化DNA分子。线性化DNA分子上基因之间的同源重组在recBC sbcA大肠杆菌菌株中创建了大型嵌合bphC基因文库。经过三轮重组后，通过三交叉很容易获得具有四部分基因片段的嵌合bphC基因。通过采用96孔微量滴定板高通量筛选，从嵌合bphC基因文库中筛选出热稳定的嵌合2,3-二羟基联苯1,2-双加氧酶。这为体内蛋白质的定向进化开辟了一条新途径。
