A Cloning and ϵ-Epitope-Tagging Insert for the Expression of Polymerase Chain Reaction-Generated cDNA Fragments in Escherichia coli and Mammalian Cells
✍ Scribed by Z. Olah; C. Lehel; G. Jakab; W.B. Anderson
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 1012 KB
- Volume
- 221
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An intercompatible gene-tagging insert sequence was designed to conveniently introduce epitope-tagged polypeptides into bacteria and mammalian cells. The presence of rare restriction enzyme sites located between the ATG codon and the sequence encoding the introduced (\boldsymbol{\epsilon})-tag creates a general cloning site which allows efficient cloning of virtually any desired cDNA fragment produced by the polymerase chain reaction (PCR). The tagging insert sequence encodes a KGFSYFGEDLMP peptide, derived from the last 12 amino acids of the protein kinase (C) epsilon gene, to serve as a C-terminal epitope tag of the expressed protein. While the insert can be readily adapted for insertion into any expression vector, this paper details the introduction and characterization of the (\epsilon)-epitope-tagging insert into the bacterial pTrcHis (A) ( (\epsilon) TrcHis (A) ) vector and into the metallothionein promoter-driven eukaryotic ( (\epsilon) MTH) expression vector. The expressed (\epsilon)-tagged proteins can be readily detected with a commercially available antibody specific for the (\epsilon)-peptide. Immunoscreening of Escherichia coli colonies transformed with the PCR-generated cDNA inserted into the (\epsilon) TrcHis A vector enables rapid, direct biochemical characterization of the PCR product. The biochemically characterized gene constructs from the (\epsilon) TreHis A plasmid can be inserted into the (\epsilon) MTH vector by a single subcloning step using the introduced compatible cohesive ends. This (\epsilon-) epitope-tagging insert provides investigators with a versatile, uncomplicated, and reliable method of expressing an epitope-tagged PCR product in the cell type of interest. The (\epsilon)-epitope tagging of the expressed peptide facilitated: (1) immunoscreening of NIH 3T3 transfectants by Western blot analysis for the intro-