An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli
Reisinger, C.; Kern, A.; Fesko, K.; Schwab, H.
Applied Microbiology and Biotechnology 77(1): 241-244
2007
ISSN/ISBN: 0175-7598
PMID: 17786428
DOI: 10.1007/s00253-007-1151-1
Accession: 015017065
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.