Jézéquel Laetitia, Loeper Jacqueline, Pompon Denis
Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Biotechniques. 2008 Nov;45(5):523-32. doi: 10.2144/000112954.
Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.
编码具有预定义交叉点的嵌合酶的组合文库是研究和模拟结构-功能关系以及基于多元统计对酶进行功能优化的有用工具。所提出的方法称为嵌合体有序文库的序列独立生成（SIGNAL），它允许在两个或多个蛋白质之间轻松改组任何预定义的氨基酸片段。该方法特别适合于蛋白质结构模块的交换。该程序也非常适合以自动化方式生成独立于序列相似性排列的组合文库。首先使用基于生物素-抗生物素蛋白的方法通过PCR从编码感兴趣酶的单链模板中提取要重组的序列片段。该技术可减少最终文库中亲本模板的污染。特定的PCR引物可扩增两个互补的嵌合DNA片段，它们在要交换的区域重叠。最后使用融合PCR将片段重新组装。通过使用这种高度模块化的方法构建一组嵌合CYP2B酶来说明该过程。
