title: A versatile and efficient high-throughput cloning tool for structural biology.
abstract: Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. e.geertsma@bioc.uzh.ch
authors.forename: Eric R
authors.initials: ER
authors.lastname: Geertsma
authors.forename: Raimund
authors.initials: R
authors.lastname: Dutzler
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001695:Biology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005240:Feasibility Studies; D026901:Membrane Transport Proteins / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics
chemical_list: D026901:Membrane Transport Proteins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific