title: Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes.
abstract: We have developed an in vitro evolution method for the selection for catalytic activity under the conditions of free intermolecular interaction between the enzyme and a substrate. The destabilized ternary enzyme-mRNA-ribosome complexes generated by a ribosome display of the mutant library are compartmentalized in vitro by forming a water-in-oil emulsion in such a way, that every droplet would on average contain no more than a single complex. After the complex dissociates within the droplet, the released enzyme molecule is free to interact with a substrate under the selection pressure on all its enzymatic properties (substrate binding, product formation, rate acceleration and turnover) simultaneously-an opportunity for the most efficient selection for catalytic activity. By using the M-MuLV reverse transcriptase as a model, we demonstrated the high efficiency of the method selecting for mutants synthesizing cDNA at increased temperature. A slightly modified compartmentalized ribosome display (CRD) could be used for the selection of other enzymes activities (e.g. DNA polymerase, RNA or DNA ligase terminal nucleotidyl transferase activity). Employment of microfluidics technique could broaden the scope of CRD technique furthermore providing an opportunity to select almost any enzyme at single molecule level under desired conditions.
authors.affiliation: Thermo Fisher Scientific, 02241 Vilnius, Lithuania.
authors.forename: Remigijus
authors.initials: R
authors.lastname: Skirgaila
authors.forename: Vaidas
authors.initials: V
authors.lastname: Pudzaitis
authors.forename: Sigitas
authors.initials: S
authors.lastname: Paliksa
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Vaitkevicius
authors.forename: Arvydas
authors.initials: A
authors.lastname: Janulaitis
mesh_terms: D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004655:Emulsions; D015723:Gene Library; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D004655:Emulsions; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase
keywords: M-MuLV reverse transcriptase; compartmentalized ribosome display; protein in vitro evolution; thermostable reverse transcriptase