Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx).
Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx).
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DOI：
10.1093/nar/gkn358
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Baldwin AJ;Busse K;Simm AM;Jones DD
三核苷酸交换（TriNEx）是一种在定向进化过程中通过将一个连续的三核苷酸序列随机替换为另一个来产生新的分子多样性的方法。使用工程转座子 MuDel 在靶基因的随机位置删除单个三核苷酸序列，随后将其替换为由称为 SubSeqNNN 的 DNA 盒提供的随机三核苷酸序列。使用编码 TEM-1 β-内酰胺酶的 bla 基因作为模型来证明 TriNEx 的有效性。序列分析显示，突变分布在整个bla中，变异体包含单、双和三核苷酸变化。由此产生的许多氨基酸取代对 TEM-1 的体内活性具有显着影响，包括对头孢他啶的活性增加高达 64 倍，对抑制剂克拉维酸的耐药性增加高达 8 倍。许多观察到的氨基酸取代只能通过每个密码子交换至少两个核苷酸来实现，包括电荷开关（R164D）和芳香族取代（W165Y）突变。因此，通过将定点饱和诱变的能力与全基因诱变的能力相结合，TriNEx 可以生成各种具有改变特性的蛋白质变体，从而在整个基因中随机引入突变。
Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeqNNN. The bla gene encoding TEM-1 β-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.
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影响因子：
5.6
作者：
Huang, WZ;Petrosino, J;Palzkill, T
通讯作者：
Palzkill, T
影响因子：
6.4
作者：
Ferraz RM;Vera A;Arís A;Villaverde A
通讯作者：
Villaverde A
在目标基因中随机位置的三重核苷酸去除：TEM-1β-内酰胺酶对氨基酸缺失的耐受性。
影响因子：
14.9
作者：
Jones, D Dafydd
通讯作者：
Jones, D Dafydd
影响因子：
46.9
作者：
Murakami, H;Hohsaka, T;Sisido, M
通讯作者：
Sisido, M
影响因子：
48
作者：
Yuen, Courtney M.;Liu, David R.
通讯作者：
Liu, David R.