A cloning and epsilon-epitope-tagging insert for the expression of polymerase chain reaction-generated cDNA fragments in Escherichia coli and mammalian cells.
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 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 KGF-SYFGEDLMP 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 TrcHis 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Oláh
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Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Lehel
C
C
Jakab
G
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Anderson
W B
WB
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics* / Q000276:immunology; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000235:genetics* / Q000276:immunology; D051744:Protein Kinase C-epsilon; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D007527:Isoenzymes; D011994:Recombinant Proteins; D008668:Metallothionein; C497256:Prkce protein, mouse; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon