Expression and purification of movement protein of Cucumber green mottle mosaic virus using a plant-virus expression system

To produce and purify movement protein of Cucumber green mottle mosaic virus (CGMMV MP), a plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used. The CGMMV MP ORF was in frame inserted between the P1 and HC-Pro ORFs of the ZYMV vector. The infectious activity of the vector was approved by rubbing the plasmid on Chenopodium quinoa leaves and observing local lesions. Individual lesions were mechanically transferred to the systemic host plant zucchini squash at the stage of two-cotyledon. The stability of recombinant protein expression was assessed by successive passages of recombinant from infected plant and throughout the period of 30 days after inoculation in a single plant and after 10 serial passages. Then, the leaves tissues of inoculated plant were analyzed by RT-PCR and western blot analysis. Recombinant protein was purified using centrifuge method combine with gel extraction; each step was sampled and analyzed by western blotting and SDS-PAGE. The results showed approximately 1.8–2.2 mg recombinant MP per 100 g tissues were purified from leaves two weeks post inoculation. Also, the vector was remarkably stable in squash after 10 serial passages and 30 days. The procedure provides a convenient and fast method for production of large quantities of pure CGMMV MP in planta.