Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.
Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.
Georgiev
O
O
Institut für Molekular Biologie II, der Unversität Zürich, Switzerland.
Bourquin
J P
JP
Gstaiger
M
M
Knoepfel
L
L
Schaffner
W
W
Hovens
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D000939:Epitopes; D005057:Eukaryotic Cells; D005822:Genetic Vectors; D008322:Mammals; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009206:Myocardium / Q000201:enzymology; D010957:Plasmids; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D000939:Epitopes; D011994:Recombinant Proteins; D011494:Protein Kinases