Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.

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.

Exploring the Conformational Landscape of Poly(l-lysine) Dendrimers Using Ion Mobility Mass Spectrometry.

Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen). Each molecule gives a highly discrete CCS distribution suggestive of single conformers for each m/z value. The DTCCSN2 values of this series of molecules (molecular weight: 330-16,214 Da) range from 182 to 2941 Å2, which spans the CCS range that would be found by many synthetic molecules including supramolecular compounds and many biopolymers. The CCS values for each charge state were highly reproducible in day-to-day analysis on each instrument, although we found small variations in the absolute CCS values between instruments. The rigidity of each dendrimer was probed using collisionally activated and high-temperature IM-MS experiments, where no evidence for a significant CCS change ensued. Taken together, this data indicates that these polymers are candidates for CCS calibration and could also help to reconcile differences found in CCS measurements on different instrument geometries.