Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris.
Tobacco 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 alpha-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 assays.
Ruiz-Ferrer
Virginia
V
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Goytia
Elisa
E
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Martínez-García
Belén
B
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
López-Abella
Dionisio
D
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
López-Moya
Juan José
JJ
Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000502:physiology; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017800:Potyvirus / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000821:virology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus