Expression of the measles virus nucleoprotein gene in Escherichia coli and assembly of nucleocapsid-like structures.
To investigate the use of fusion systems to aid the purification of recombinant proteins for structure/function studies and potential uses as diagnostic reagents, the measles virus (MV) gene encoding the nucleoprotein was cloned and expressed in Escherichia coli in three forms: as a full-length intact protein and as two fusion proteins. Expression of the intact N gene under the control of the tac promoter in the pTrc99c plasmid produced a protein of the correct size (60 kDa) which represented approx. 4% of the total cellular protein, and was recognised by known measles positive human sera. 'Herringbone' structures characteristic of paramyxovirus nucleocapsids (NuC) were identified in fractured cells examined by electron microscopy. The production of NuC-like structures in a prokaryotic cell indicates folding of the nucleoprotein can occur in the absence of MV genomic RNA, other MV-encoded gene products and eukaryotic cell proteins or RNA, to produce structures which are morphologically and antigenically similar to those seen in virus-infected cells. Conversely, synthesis of N protein as a fusion protein with either E. coli beta-galactosidase or the E. coli maltose-binding protein resulted in the production of fused proteins which could not be assembled into NuC-like structures or readily used as diagnostic reagents. However, the ability of MV N protein to form NuC-like structures in E. coli will facilitate structure/function and mutational analysis of the NuC protein.
Warnes
A
A
Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK.
Fooks
A R
AR
Dowsett
A B
AB
Wilkinson
G W
GW
Stephenson
J R
JR
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D015870:Gene Expression; D058619:Maltose-Binding Proteins; D008459:Measles virus / Q000235:genetics* / Q000276:immunology; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins; D009024:Morphogenesis; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; C058314:nucleoprotein, Measles virus; D001616:beta-Galactosidase