Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris

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Abstract
9 páginas, 3 figuras, 1 tabla -- PAGS NROS. 241-249Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae α-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assaysThis work was supported by grants BIO2000-0914, BIO 2000-1605-C02-02 and AGL2001-2141 from CICYT (Spain), and 07M-0123-2000 and 07M-0072-2002 from Comunidad de Madrid (Spain). V. R.-F. and E. G. are recipients of PNFPI fellowships from MCyT (Spain), and B. M.-G. is the recipient of a post-doctoral contract from the Comunidad de Madrid (Spain).Peer reviewe