Synthesis of the measles virus nucleoprotein in yeast Pichia pastoris and Saccharomyces cerevisiae.
The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology. The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively. Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P. pastoris and S. cerevisiae, respectively. This protein is one of most abundantly expressed in yeast. After purification nucleocapsid-like particles (NLPs) derived from both yeast genera appeared to be similar to those detected in mammalian cells infected with measles virus. A spontaneous assembly of nucleoprotein into nucleocapsid-like particles in the absence of the viral leader RNA or viral proteins has been shown. Compartmentalisation of recombinant protein into large compact inclusions in the cytoplasm of yeast S. cerevisiae by green fluorescence protein (GFP) fusion has been demonstrated. Sera from measles patients reacted with the recombinant protein expressed in both yeast genera and a simple diagnostic assay to detect measles IgM could be designed on this basis.
Slibinskas
Rimantas
R
Institute of Biotechnology, Vilnius LT-2028, Graiciuno, Lithuania.
Samuel
Dhanraj
D
Gedvilaite
Alma
A
Staniulis
Juozas
J
Sasnauskas
Kestutis
K
D015153:Blotting, Western; D002451:Cell Compartmentation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood; D007700:Kinetics; D008164:Luminescent Proteins; D008457:Measles / Q000821:virology; D008459:Measles virus / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D009698:Nucleoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D010843:Pichia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D007075:Immunoglobulin M; D008164:Luminescent Proteins; D009698:Nucleoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins