title: Constructing high complexity synthetic libraries of long ORFs using in vitro selection.
abstract: We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly 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 frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel.
authors.affiliation: Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: G
authors.initials: G
authors.lastname: Cho
authors.forename: A D
authors.initials: AD
authors.lastname: Keefe
authors.forename: R
authors.initials: R
authors.lastname: Liu
authors.forename: D S
authors.initials: DS
authors.lastname: Wilson
authors.forename: J W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D016368:Frameshift Mutation / Q000235:genetics; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D019151:Peptide Library; D014176:Protein Biosynthesis / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012641:Selection, Genetic; D016944:Sulfolobus; D013698:Templates, Genetic
chemical_list: D003062:Codon; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D019151:Peptide Library; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D007209:Indole-3-Glycerol-Phosphate Synthase; D011088:DNA Ligases