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 >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 p62^TCF 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.