Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.
Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.
Juozapaitis
Mindaugas
M
Institute of Biotechnology, V. Graiciuno 8, Vilnius LT-02241, Lithuania.
Serva
Andrius
A
Zvirbliene
Aurelija
A
Slibinskas
Rimantas
R
Staniulis
Juozas
J
Sasnauskas
Kestutis
K
Shiell
Brian J
BJ
Wang
Lin-Fa
LF
Michalski
Wojtek P
WP
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002499:Centrifugation, Density Gradient; D002685:Chiroptera; D003001:Cloning, Molecular; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D045403:Henipavirus / Q000235:genetics* / Q000276:immunology; D045464:Henipavirus Infections / Q000276:immunology; D006736:Horses; D006801:Humans; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013552:Swine; D022701:Virosomes / Q000648:ultrastructure
D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D022701:Virosomes