title: Recombinant cloning strategies for protein expression.
abstract: A variety of methods to create specific constructs for protein expression, in a broad range of organisms, are available nowadays. Restriction enzyme-free, ligation-independent and recombinase-based cloning methods have enabled high-throughput protein expression for structural and functional studies. These methods are also instrumental for modification of target genes including gene truncations, site-specific mutagenesis and domain swapping. Here, we describe the most common cloning techniques that are currently at hand for recombinant protein expression studies, including a brief overview of techniques associated with co-expression experiments. We also provide an inventory of many of the available reagents for the various cloning methods, and an overview for some computational tools that can help with the design of expression constructs.
authors.affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. Electronic address: p.celie@nki.nl.
authors.forename: Patrick Hn
authors.initials: PH
authors.lastname: Celie
authors.affiliation: European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Annabel Ha
authors.initials: AH
authors.lastname: Parret
authors.affiliation: Division of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
authors.forename: Anastassis
authors.initials: A
authors.lastname: Perrakis
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes