An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli.
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.
Reisinger
Christoph
C
Research Centre Applied Biocatalysis, Petersgasse 14/4, Graz, Austria. christoph.reisinger@a-b.at
Kern
Alexander
A
Fesko
Kateryna
K
Schwab
Helmut
H
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