title: Cell-free protein expression and functional assay in nanowell chip format.
abstract: The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.
authors.affiliation: Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. angenend@molgen.mpg.de
authors.forename: Philipp
authors.initials: P
authors.lastname: Angenendt
authors.forename: Lajos
authors.initials: L
authors.lastname: Nyarsik
authors.forename: Witold
authors.initials: W
authors.lastname: Szaflarski
authors.forename: Jörn
authors.initials: J
authors.lastname: Glökler
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
authors.forename: Hans
authors.initials: H
authors.lastname: Lehrach
authors.forename: Dolores J
authors.initials: DJ
authors.lastname: Cahill
authors.forename: Angelika
authors.initials: A
authors.lastname: Lueking
mesh_terms: D002474:Cell-Free System; D049452:Green Fluorescent Proteins / Q000378:metabolism; D036103:Nanotechnology; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase