Histidine-tagging and purification of tobacco etch potyvirus helper component protein

The coding sequence for a series of six histidines this-tag) was inserted n ear the 5' terminus of the helper component (HC) coding region of tobacco e tch potyvirus (TEV). Full length genomic clones containing the his-tag codi ng sequence were infectious and produced symptoms in tobacco (Nicotiana tab acum) similar to those induced by wild-type TEV. The modified virus was gen etically stable and the his-tag sequence was maintained through at least fo ur cycles of aphid transmission. A protocol for purifying rapidly the HC pr otein, based on the affinity of its his-tag for Ni2 +-charged resin, yielde d large amounts of his-tagged HC protein that was fully functional as demon strated by aphid transmission experiments. (C) 1999 Elsevier Science B.V. A ll rights reserved.