Constructing high complexity synthetic libraries of long ORFs using in vitro selection

We present a method that can significantly increase the complexity of prote in libraries used for in vitro or in vivo protein selection experiments. Pr otein libraries are often encoded by chemically synthesized DNA, in which p art of the open reading frame is randomized. There are, however, major obst acles associated with the chemical synthesis of long open reading frames, e specially those containing random segments. Insertions and deletions that o ccur during chemical synthesis cause frameshifts, and stop codons in the ra ndom region will cause premature termination. These problems can together g reatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshi fts and stop codons. These smaller segments are then ligated together to fo rm combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libra ries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to gener ate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensi ty for forming amphipathic cc-helical or P-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. (C) 2000 Academic Press.