Characterization and diagnostic potential of foreign epitope-presenting Ty1 virus-like particles expressed in Escherichia coli and Pichia pastoris.
We expressed a truncated p1 protein (p1-379) from the Saccharomyces cerevisiae retrotransposon Ty1 in the cytosol of Escherichia coli and Pichia pastoris, achieving maximum expression levels of 20 and 65 mg/l, respectively. Two well-characterized epitopes from beet necrotic yellow vein virus (BNYVV) were used to evaluate the virus-like particles (VLPs) as a presentation system for synthetic antigens. The epitopes were placed near the externally located N-terminus and at the internally located C-terminus of the p1 protein. Electron microscopy showed all particles to be morphologically similar to wild-type Ty1-VLPs from S. cerevisiae. However, fewer VLPs were observed in P. pastoris, suggesting that posttranslational modifications might inhibit binding to the carbon-coated electron microscopy grids. BNYVV epitopes were detected with specific monoclonal antibodies by Western blot and ELISA, with a detection limit as low as 5 ng/ml. The VLPs are therefore promising candidates for diagnostic standards and future vaccine development.
Uhde-Holzem
Kerstin
K
Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
Fischer
Rainer
R
Commandeur
Ulrich
U
D000941:Antigens / Q000096:biosynthesis / Q000235:genetics; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008854:Microscopy, Electron; D010843:Pichia / Q000235:genetics*; D010942:Plant Viruses / Q000235:genetics* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018626:Retroelements; D022701:Virosomes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D000941:Antigens; D000939:Epitopes; D011994:Recombinant Proteins; D018626:Retroelements; D022701:Virosomes