Purification and analysis of polyhistidine-tagged human parvovirus B19 VP1 and VP2 expressed in insect cells.
Human parvovirus B19 is an autonomously replicating human pathogen with a specific tropism for human erythroid progenitor cells. There is an interest in producing empty nucleocapsids of B19 as they can be used as tools in molecular biology and diagnostics. Native B19 virus particles are formed from two structural viral proteins, VP1 and VP2. The VP2 protein alone is able to self assemble and consequently form virus-like particles (VLPs) in heterologous expression systems. Purification of recombinant VLPs has been conducted using various traditional methods. These include laborious and time-consuming, e.g. cesium chloride or sucrose gradient ultracentrifugation steps, allowing limited working volumes to be processed. Therefore, an alternative purification method enabling process scale-up was developed and evaluated. Polyhistidine-tagged versions of B19 VP1 and VP2 capsid proteins were engineered and produced using the baculovirus expression system. The recombinant protein products were purified by immobilized metal-ion affinity chromatography (IMAC) and analyzed by SDS-PAGE, immunoblotting, electron microscopy, and enzyme-linked immunosorbent assays. Further, the immunological properties of the recombinant proteins were evaluated. The results showed that the VP2 fusion protein assembled into capsid-like structures and that both VP1 and VP2 following purification by IMAC have potential as antigens for diagnosis of a B19 infection.
Michel
Patrik O
PO
Nanoscience Center, Department of Biological and Environmental Science, University of Jyväskylä, FIN-40014 Jyväskylä, Finland. patrik.michel@jyu.fi
Mäkelä
Anna R
AR
Korhonen
Eila
E
Toivola
Jouni
J
Hedman
Lea
L
Söderlund-Venermo
Maria
M
Hedman
Klaus
K
Oker-Blom
Christian
C
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D015672:Erythroid Precursor Cells; D005786:Gene Expression Regulation; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000166:cytology*; D016732:Parvovirus B19, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000378:metabolism*
D036022:Capsid Proteins; D011994:Recombinant Proteins; C095547:capsid protein VP1, parvovirus B19; C095548:capsid protein VP2, parvovirus B19; C033223:polyhistidine; D006639:Histidine