efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli

• Published in: Applied microbiology and biotechnology 2007-11, Vol.77 (1), p.241-244
• Main Authors: Reisinger, Christoph, Kern, Alexander, Fesko, Kateryna, Schwab, Helmut
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cloning; Cloning, Molecular - methods; DNA polymerase; E coli; Editing; Enzymes; Escherichia coli - genetics; Expression plasmid; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Enzymologic; Genes; Genetic engineering; Genetic technics; Genetic Vectors - genetics; Glycine Hydroxymethyltransferase - genetics; Glycine Hydroxymethyltransferase - metabolism; Methods. Procedures. Technologies; Models, Genetic; Parallel cloning; PCR-cloning; Plasmids; Plasmids - genetics; Polymerase chain reaction; Polymerase Chain Reaction - methods; Protein expression; Proteins; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant protein expression; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Studies; TA-cloning; Vectors (Biology); Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-007-1151-1

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.