Histidine-tagging and purification of tobacco etch potyvirus helper component protein.
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 tabacuma) 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 Ni(2+)-charged resin, yielded large amounts of his-tagged HC protein that was fully functional as demonstrated by aphid transmission experiments.
Department of Plant Pathology, University of Kentucky, Lexington 40546, USA.
S
S
Blanc
V V
VV
Dolja
C
C
Llave
T P
TP
Pirone
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000821:virology; D001483:Base Sequence; D006639:Histidine / Q000378:metabolism*; D007303:Insect Vectors / Q000821:virology; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology; D010947:Plants, Toxic; D017800:Potyvirus / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D014026:Nicotiana / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D017361:Viral Nonstructural Proteins; D006639:Histidine