title: A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.
abstract: A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.
authors.affiliation: Department Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany; Department Molecular Genetics and Preparative Molecular Biology, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Blagovesta
authors.initials: B
authors.lastname: Popova
authors.affiliation: Department Molecular Genetics and Preparative Molecular Biology, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany; Department Dermatology, Venereology and Allergology, University Medical Center, Göttingen, Germany; Information Network of Departments of Dermatology (IVDK), Göttingen, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schubert
authors.affiliation: Theoretical Biology and Biophysics, Group T-6, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America; Institute for Mathematics and Informatics, Universität Greifswald, Greifswald, Germany; Department Bioinformatics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Bulla
authors.affiliation: Department Bioinformatics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany; Neurobiology Laboratory, German Primate Center GmbH, Göttingen, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Buchwald
authors.affiliation: Department Molecular Genetics and Preparative Molecular Biology, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany; Department Molecular Genetics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Kramer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D015723:Gene Library; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D012015:Reference Standards; D012984:Software; D020124:Thermotoga maritima / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins