A combinatorial approach to hybrid enzymes independent of DNA homology
A combinatorial approach to hybrid enzymes independent of DNA homology
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DOI：
10.1038/70754
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发表时间：
1999-12-01
影响因子：
46.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Ostermeier, M;Shim, JH;Benkovic, SJ
我们提出了一种方法，称为增量截短杂交酶（ITCHY）的创建，创建基因之间的组合融合库的方式是独立的DNA同源性。我们比较了ITCHY和DNA改组的能力，以创建种间融合的大肠杆菌和人类甘氨酰胺核糖核苷酸转化酶基因，只有50%的DNA水平上的同一性的片段之间的库。从每个文库中随机选择的几个阳性序列的测序表明，ITCHY鉴定了一组更多样化的活性融合点，包括非同源区域中的融合点和与序列比对不同的交叉点。此外，ITCHY发现的一些在非同源位置融合的杂交体的活性大于或等于通过DNA改组发现的杂交体的活性。
We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.