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. 1991 Oct 15;88(20):8972–8976. doi: 10.1073/pnas.88.20.8972

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

R Janknecht 1, G de Martynoff 1, J Lou 1, R A Hipskind 1, A Nordheim 1, H G Stunnenberg 1
PMCID: PMC52633  PMID: 1924358

Abstract

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.

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These references are in PubMed. This may not be the complete list of references from this article.

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