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Keywords:

  • binary vector;
  • Cowpea mosaic virus;
  • GATEWAY;
  • molecular farming;
  • transient expression

Summary

Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5′-untranslated region (UTR) and the 3′-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [Sainsbury, F. and Lomonossoff, G.P. (2008)Plant Physiol. 148, 1212–1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.