RAPID EVOLUTION OF A PROTEIN IN-VITRO BY DNA SHUFFLING
RAPID EVOLUTION OF A PROTEIN IN-VITRO BY DNA SHUFFLING
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DOI：
10.1038/370389a0
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发表时间：
1994-08-04
期刊：
影响因子：
64.8
通讯作者：
STEMMER, WPC
中科院分区：
文献类型：
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作者：
STEMMER, WPC
DNA 改组是一种通过随机片段化和聚合酶链式反应 (PCR) 重组对所选突变基因池进行体外同源重组的方法 (1)。称为遗传算法 (2-4) 的计算机模拟已经证明了迭代同源重组对于序列进化的重要性。寡核苷酸盒诱变(5-11) 和易错PCR 不是组合的，因此在搜索序列空间(1,14) 方面受到限制。我们已经在 β-内酰胺酶模型系统 (9,19) 中测试了用于分子进化 (14-18) 的诱变 DNA 改组。三个周期的改组和两个与野生型 DNA 的回交周期，以消除非必需突变，然后分别选择浓度逐渐增加的抗生素头孢噻肟。我们在此报告，选定的突变体的最低抑制浓度为 640 mu g ml(-1)，比任何已发表的 TEM-1 衍生酶增加了 32,000 倍和 64 倍。盒式诱变和易错 PCR 只导致了 16 倍的增加(9)。
DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly(1). Computer simulations called genetic algorithms(2-4) have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis(5-11) and error-prone PCR are not combinatorial and thus are limited in searching sequence space(1,14). We have tested mutagenic DNA shuffling for molecular evolution(14-18) in a beta-lactamase model system(9,19). Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 mu g ml(-1), a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase(9).