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

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Abstract

The coding sequence for a series of six histidines (his-tag) was inserted near the 5′ terminus of the helper component (HC) coding region of tobacco etch potyvirus (TEV). Full length genomic clones containing the his-tag coding sequence were infectious and produced symptoms in tobacco (Nicotiana tabacum) similar to those induced by wild-type TEV. The modified virus was genetically stable and the his-tag sequence was maintained through at least four cycles of aphid transmission. A protocol for purifying rapidly the HC protein, based on the affinity of its his-tag for Ni2+-charged resin, yielded large amounts of his-tagged HC protein that was fully functional as demonstrated by aphid transmission experiments.

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Acknowledgements

Research supported in part by funds from the University of Kentucky THRI. C.L. was the recipient of a fellowship from the PFPI and of financial support from CICYT (Pb94-0023) and CAM (07B-0024-1997) (Spain). We thank David Thornbury for valuable discussions and Jannine Baker for excellent technical assistance.

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Present address: Station de Pathologie Comparée, INRA-CNRS, 30 380 Saint Christol-les-Alès, France.

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