Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.
Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.
Institute for Molecular Biology, Hannover Medical School, Federal Republic of Germany.
R
R
Janknecht
G
G
de Martynoff
J
J
Lou
R A
RA
Hipskind
A
A
Nordheim
H G
HG
Stunnenberg
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D006367:HeLa Cells; D006639:Histidine; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification; D012516:Osteosarcoma; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D011995:Recombination, Genetic; D026362:Serum Response Factor; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics*
D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D006639:Histidine