Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli

Brudenell, E. L.; Pohare, M. B.; Zafred, D.; Phipps, J.; Hornsby, H. R.; Darby, J.; Dai, J.; Liggett, E.; Cain, K.; Barran, P. E.; de Silva, T. I.; Sayers, J. R.

doi:10.1101/2024.01.08.574531

bioRxiv · 10.1101/2024.01.08.574531

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli

Brudenell, E. L.·Pohare, M. B.·Zafred, D.·Phipps, J.·Hornsby, H. R.·Darby, J.·Dai, J.·Liggett, E.·Cain, K.·Barran, P. E.·de Silva, T. I.·Sayers, J. R.

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Abstract

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

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Brudenell, E. L., Pohare, M. B., Zafred, D., Phipps, J., Hornsby, H. R., Darby, J., Dai, J., Liggett, E., Cain, K., Barran, P. E., de Silva, T. I., Sayers, J. R.. 2024-01-09. Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli. https://doi.org/10.1101/2024.01.08.574531

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