Expression of Lassa virus nucleocapsid protein segments in bacteria: purification of high-level expression products and their application in antibody detection.
The Lassa virus nucleocapsid protein gene and segments from it were expressed in Escherichia coli under the control of the lac promoter in pUC-based plasmids. Expression of the near full-length protein [amino acid (aa) residues 12-570] fused to an N-terminal sequence of vector-derived 6 aa was not particularly efficient, and neither was that of a smaller N-terminal segment (aa 6-201) which was also fused at its C terminus to the remainder of the lacZ gene product. By contrast, the C-terminal 370 aa could be expressed at levels approaching 10% of total cellular protein. All the recombinant proteins were associated with the insoluble fraction after sonication of the bacteria. The inefficiently expressed products did not appear to be any more susceptible to proteolytic degradation. The distribution of codons rarely used in E. coli genes was relatively uniform along the nucleocapsid gene sequence. These results are consistent with the regulation of transcriptional or translational efficiency by features of the sequence downstream from the promoter and ribosome-binding site. The C-terminal segment (aa 201-570 representing 65% of the authentic protein) was purified by ion exchange chromatography and shown to be active when used as antigen in enzyme-linked immunoassays for virus-specific antibodies.
Barber
G N
GN
Special Pathogens Reference Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
Clegg
J C
JC
Chamberlain
J
J
D000914:Antibodies, Viral / Q000032:analysis; D001116:Arenaviridae / Q000235:genetics*; D002213:Capsid / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005814:Genes, Viral; D007836:Lassa virus / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins