An effective family shuffling method using single-stranded DNA.
An effective family shuffling method using single-stranded DNA.
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DOI：
10.1016/s0378-1119(99)00547-8
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发表时间：
2000-02
期刊：
影响因子：
3.5
通讯作者：
Miho Kikuchi;Kouhei Ohnishi;S. Harayama
中科院分区：
文献类型：
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作者：
Miho Kikuchi;Kouhei Ohnishi;S. Harayama
家族改组是体外蛋白质进化中最强大的技术之一，它总是涉及将基因片段重组成亲本基因序列的问题，因为这样的过程会阻止嵌合序列的形成。为了提高家族改组中杂交体形成的效率，使用单链DNA（ssDNAs）作为模板。制备了两个儿茶酚2，3-双加氧酶基因nahH和xylE的ssDNA，xylE链与nahH链互补。当这些ssDNA被DNase I消化并重组时，嵌合基因的获得率为14%，这比用双链DNA改组获得的不到1%的比率高得多。通过这种基于ssDNA的DNA改组获得了比亲本酶XylE和NahH更热稳定的嵌合儿茶酚2，3-双加氧酶。
Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fragments into parental gene sequences, because such a process prevents the formation of chimeric sequences. In order to improve the efficiency of hybrid formation in family shuffling, single-stranded DNAs (ssDNAs) were used as templates. The ssDNAs of two catechol 2,3-dioxygenase genes, nahH and xylE, were prepared, the xylE strand being complementary to the nahH strand. When these ssDNAs were digested by DNase I and reassembled, chimeric genes were obtained at a rate of 14%, which was much higher than the rate of less than 1% obtained by shuffling with double-stranded DNAs. Chimeric catechol 2,3-dioxygenases that were more thermally stable than the parental enzymes, XylE and NahH, were obtained by this ssDNA-based DNA shuffling.