Growth factor engineering by degenerate homoduplex gene family recombination
Growth factor engineering by degenerate homoduplex gene family recombination
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DOI：
10.1038/nbt757
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发表时间：
2002-12-01
影响因子：
46.9
通讯作者：
Monticello, DJ
中科院分区：
文献类型：
--
作者：
Coco, WM;Encell, LP;Monticello, DJ
人们对工程改造人生长因子作为潜在的治疗激动剂和拮抗剂有很大的兴趣。我们用合成DNA重组方法接近这个目标。我们使用多个多态性“支架”寡核苷酸将来自编码表皮生长因子（EGF）的哺乳动物基因的多态性并入“顶链”寡核苷酸池。然后通过缺口填充和连接来连接顶部链。这种方法避免了高度简并寡核苷酸连接中的异源双链体退火，从而实现了完全随机重组。从人-小鼠嵌合文库克隆的基因捕获了亲本多态性的每一种可能的排列，产生了一个明显完整的重组基因家族文库，这在以前没有描述过。该文库产生了一种嵌合蛋白，其激动剂活性增强了123倍。第二个库从五个哺乳动物EGF同源物具有最高的重组密度（1个交换每12.4 bp）。五个同源物库产生了迄今报道的最强结合的hEGF变体。此外，它含有具有拮抗剂性质的强结合EGF变体。我们对DNA改组的较少偏见的方法应该对各种蛋白质的工程有用。
There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human-mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.