Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.
A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.
Dolja
V V
VV
Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331, USA. doljav@bcc.orst.edu
Peremyslov
V V
VV
Keller
K E
KE
Martin
R R
RR
Hong
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000302:isolation & purification; D017790:Closterovirus / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D010944:Plants / Q000821:virology*; D017800:Potyvirus / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine