Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus

Ralf Janknecht, Guy De Martynoff, Jueren Lou, Robert A. Hipskind, Alfred Nordheim, Hendrik G. Stunnenberg

Onderzoeksoutput: Bijdrage aan tijdschriftArtikelpeer review

441 Citaten (Scopus)

Samenvatting

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 >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.

Originele taal-2Engels
Pagina's (van-tot)8972-8976
Aantal pagina's5
TijdschriftProceedings of the National Academy of Sciences of the United States of America
Volume88
Nummer van het tijdschrift20
DOI's
StatusGepubliceerd - 15 okt. 1991
Extern gepubliceerdJa

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