Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling
Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling
复制标题
DOI：
10.1093/protein/15.10.843
复制
发表时间：
2002-10-01
期刊：
影响因子：
--
通讯作者：
Nishigaki, K
中科院分区：
文献类型：
--
作者：
Kitamura, K;Kinoshita, Y;Nishigaki, K
进化蛋白质工程目前正进入一个新的阶段，除了传统的点突变之外，还需要新的技术来产生蛋白质文库。在这种情况下，改组和重排DNA块（导致蛋白质库）的新方法的报告。设计了一个用于产生组合多样性的过程循环，并将其命名为基于Y-连接的区块改组（YLBS）。通过将其应用于模块大小和氨基酸大小的块的改组，进行了方法上的改进。用模块大小的GFP块运行三个循环的YLBS导致八块改组文库的高多样性。进行GFP的中心四个块的部分改组以获得有效改组的蛋白质，导致完整排列。还通过YLBS进行了氨基酸单体大小的块的改组，获得了超过10个改组分子的多样性。在这些实验中遇到的缺失问题被证明可以通过驯服IIS型限制性内切酶的额外措施来解决。每个块的出现频率是偏斜的，但在允许的范围内。因此，YLBS是第一种产生大量不同的改组蛋白质，轻松重组结构域，外显子和模块的通用方法。
Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method for shuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10(10) shuffled molecules was attained. The deletion problems encountered during these experiments were shown to be solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.