Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells

Abstract

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.

Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes

“…3) because NP may form aggregates before binding all the R N A at higher concentrations. Harley et al (1990) reported that NP can be purified from an NP-expressing E. coli strain; the binding activity of NP with R N A was analysed by the filter binding assay and suggested that NP is self- aggregating. Although the NP used is only 50 to 60% pure, we did not assess contamination by an E. coli RNAbinding protein because our aim was to use NP only to protect RNA molecules against ribonucleases during transfection.…”