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.
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Acknowledgment
We are grateful to our cooperation partners at the DSM Research B.V., ASC&D (6160 MD Geleen, The Netherlands), for their scientific promotion. We gladly acknowledge the financial support given by the Österreichische Forschungsförderungsgesellschaft (FFG), the Province of Styria, the Styrian Business Promotion Agency (SFG), and DSM Netherlands.
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Reisinger, C., Kern, A., Fesko, K. et al. An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli . Appl Microbiol Biotechnol 77, 241–244 (2007). https://doi.org/10.1007/s00253-007-1151-1
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DOI: https://doi.org/10.1007/s00253-007-1151-1