Generation of Sendai virus nucleocapsid-like particles in yeast.
The gene encoding Sendai virus nucleocapsid protein was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. The high level of recombinant Sendai virus nucleocapsid protein expression (12-14 mg/l of yeast culture) was obtained. The evaluation of recombinant proteins expression in yeast by Western blot analysis revealed specific reactivity with immune sera. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid protein. These structures contained host RNA, which was resistant to an RNase treatment. The nucleocapsid protein revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. The development of a simple, efficient and cost-effective system for generation of Sendai virus nucleocapsid protein might help to upgrade reagents for virus serology, and facilitate investigation of virus replication and RNA encapsidation mechanisms.
Juozapaitis
Mindaugas
M
Institute of Biotechnology, V.Graiciuno 8, Vilnius LT-2028, Lithuania. jmind@ibt.lt
Slibinskas
Rimantas
R
Staniulis
Juozas
J
Sakaguchi
Takemasa
T
Sasnauskas
Kestutis
K
D003001:Cloning, Molecular; D005814:Genes, Viral; D046911:Macromolecular Substances / Q000378:metabolism; D008854:Microscopy, Electron; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D012331:RNA, Fungal / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029082:Sendai virus / Q000235:genetics*; D019065:Virus Assembly
D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; D012331:RNA, Fungal; D011994:Recombinant Proteins