title: Sequence-independent construction of ordered combinatorial libraries with predefined crossover points.
abstract: Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.
authors.affiliation: Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Jézéquel
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Loeper
authors.forename: Denis
authors.initials: D
authors.lastname: Pompon
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D019921:Biotinylation; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D032621:DNA Shuffling; D004285:Dogs; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016366:Open Reading Frames; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D004247:DNA; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); D010089:Oxidoreductases, N-Demethylating