title: An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli.
abstract: The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the "T-vector" can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3'-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5'-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.
authors.affiliation: Research Centre Applied Biocatalysis, Petersgasse 14/4, Graz, Austria. christoph.reisinger@a-b.at
authors.forename: Christoph
authors.initials: C
authors.lastname: Reisinger
authors.forename: Alexander
authors.initials: A
authors.lastname: Kern
authors.forename: Kateryna
authors.initials: K
authors.lastname: Fesko
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics*; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics