An efficient single pot DNA recombination method for protein library generation.

A simple single pot method was developed to generate a library of protein hybrids/chimeras from unrelated parents. This method allows unbiased facile DNA shuffling based recombination between the parents with the introduction of type IIB restriction enzyme, BsaXI, at multiple distinct crossover sites. To assess this methodology, B1 immunoglobulin domain of protein G and top7 were recombined at four sites, yielding 2 (32) possible hybrid proteins. Tandem colony multiplex PCR based screening was followed to screen the hybrid clones and, sequencing analysis confirmed seven hybrid clones with no sequence bias.