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

Abstract

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.

GFPs of insertion mutation generated by molecular size‐altering block shuffling

“…There have already appeared several technologies in this context: 'micro gene' methods which random-assembles DNA fragments [7], systemic circular permutation of proteins [8], random multirecombinant polymerase chain reaction (RM-PCR) [9] and incremental truncation for creating hybrid enzymes [10]. Recently, we developed the Y-ligation-based block shu¥ing (YLBS) method for the purpose of general recombination [11].…”

Towards the Creation of Novel Proteins by Block Shuffling

“…Equal amounts of the 36 dimer templates (each containing two building blocks) and 12 dimer templates containing 5' or 3' consensus sequences were mixed in the reaction mixture to obtain a library in which every position of the block sequences generated has an equal probability of encoding each of the six building blocks. Our previous study showed that many block sequences in which six building blocks were arranged into different orders were successfully constructed using this procedure [48]. Further, recently we confirmed that RM-PCR successfully combined 13 building blocks with different lengths, taken from different proteins.…”

“…To realize this concept it was necessary to develop a novel strategy capable of combining different DNA fragments without homologous sequences. We developed random multi-recombinant PCR (RM-PCR) [48,49] based upon overlap extension PCR [50]. Fig.…”

Shuffling of DNA Cassettes in a Synthetic Integron

“…However the efficient random recombination of heterologuous sequences largely remains a challenge. It has been achieved in vitro through various techniques of randomized assembly ligation and overlap extension PCR (1)(2)(3)(4)(5). However these techniques are limited in the size and number of elements they can combine.…”