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 f rom plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of h istidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterov irus (BYV). Approximately 4 mu g/g of 20-kDa BYV protein was readily isolat ed from plants systemically infected by hybrid TEV. In contrast, only minut e 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 wit h TEV HC-Pro stabilized the histidine;tagged BYV CP and facilitated purific ation of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant protei ns were recognized by histidine-tag-specific monoclonal antibody in immunob lot 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. (C) 1998 Academic Press.