Random multi-recombinant PCR for the construction of combinatorial protein libraries

摘要

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a 'random shuffling library' RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the 'alternative splicing library', RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.