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

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.

Feasibility Study To Assess The Delivery of A Novel Isometric Exercise Intervention For People With Stage 1 Hypertension In The NHS: Protocol For The Isofit-BP Study Including Amendments To Mitigate The Risk of COVID-19 (preprint)

Background: Hypertension (HTN) affects approximately 25% of the UK population and is a leading cause of mortality. Associated annual health care costs run into billions. National treatment guidance includes initial lifestyle advice, followed by anti-hypertensive medication if blood pressure (BP) remains high. However, adoption and adherence to recommended exercise guidelines, dietary advice and anti-hypertensive medication is poor. Four short bouts of isometric exercise (IE) performed 3 days per week (d/wk) at home elicits clinically significant reductions in BP in those with normal to high-normal BP. This study will determine the feasibility of delivering personalised IE to patients with Stage 1 hypertension for whom lifestyle changes would be recommended before medication within NHS primary care. Methods: : This is a randomised controlled feasibility study. Participants 18+ years, with Stage 1 hypertension, not on anti-hypertensive medication and without significant medical contraindications. Trial arms will be standard lifestyle advice (control) or isometric wall squat exercise and standard lifestyle advice. Primary outcomes include success of intervention delivery and change in BP. Secondary outcomes include accuracy of protocol delivery, execution of and adherence to protocol, recruitment rate, attrition, perception of intervention viability, cost, participant experience, and accuracy of home BP. The study will last 18 months. Sample size of 100 participants (50 per arm) allows for 20% attrition and 6.5% incomplete data, based upon 74 (37 each arm) participants (two-sided 95% confidence interval, width of 1.33 and standard deviation of 4) completing 4 weeks. Ethical approval IRAS ID: 274676. Discussion: Before the efficacy of this novel intervention to treat Stage 1 hypertension can be investigated in any large randomised controlled trial, it is necessary to ascertain if it can be delivered and carried out in a NHS primary care setting. Findings could support IE viability as a prophylactic / alternative treatment option. Trial Registration: International Standard Randomised Controlled Trial Number (ISRCTN) 13472393 registered 18 August 2020. http://www.isrctn.com/ISRCTN13472393