Kolkman J A, Stemmer W P
Maxygen Inc., 515 Galveston Drive, Redwood City, CA 94063, USA.
Nat Biotechnol. 2001 May;19(5):423-8. doi: 10.1038/88084.
Evolution of eukaryotes is mediated by sexual recombination of parental genomes. Crossovers occur in random, but homologous, positions at a frequency that depends on DNA length. As exons occupy only 1% of the human genome and introns about 24%, by far most of the crossovers occur between exons, rather than inside. The natural process of creating new combinations of exons by intronic recombination is called exon shuffling. Our group is developing in vitro formats for exon shuffling and applying these to the directed evolution of proteins. Based on the splice frame junctions, nine classes of exons and three classes of introns can be distinguished. Splice frame diagrams of natural genes show how the splice frame rules govern exon shuffling. Here, we review various approaches to constructing libraries of exon-shuffled genes. For example, exon shuffling of human pharmaceutical proteins can generate libraries in which all of the sequences are fully human, without the point mutations that raise concerns about immunogenicity.
真核生物的进化是由亲本基因组的有性重组介导的。交叉发生在随机但同源的位置，其频率取决于DNA长度。由于外显子仅占人类基因组的1%，内含子约占24%，到目前为止，大多数交叉发生在外显子之间，而不是在内含子内部。通过内含子重组产生外显子新组合的自然过程称为外显子洗牌。我们小组正在开发用于外显子洗牌的体外形式，并将其应用于蛋白质的定向进化。根据剪接框连接，可以区分九类外显子和三类内含子。天然基因的剪接框图显示了剪接框规则如何控制外显子洗牌。在这里，我们回顾了构建外显子洗牌基因文库的各种方法。例如，人类药用蛋白质的外显子洗牌可以产生文库，其中所有序列都是完全人类的，没有引起免疫原性担忧的点突变。
