Effect of an N-terminal tag on the production of SARS-CoV-2 spike protein receptor binding domain in Nicotiana tabacum Bright Yellow 2 cells

Details

Supervisors

Chaumont, François ; Peeters, Marie

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus Disease 19 (COVID-19), which started a pandemic in 2019. The SARS-CoV-2 virion is composed of four structural proteins, namely the Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins. The S protein is a heavily glycosylated trimer, with 66 N-glycosylation sites, that binds to the human angiotensin-converting enzyme 2 (hACE2) receptor at the host cell surface and enables viral entry by acting as a class I membrane fusion protein. More specifically, the binding occurs through the Receptor Binding Domain (RBD). The S protein, in particular the RBD, is the main target of host neutralising antibodies. As a result, it has been extensively studied to better understand its properties and develop vaccines. Recombinant vaccines, focused on the S protein or its RBD, were among the various strategies considered for vaccine development. Among the different recombinant protein production platforms, plants have gained increasing interest in recent years. They offer advantages compared to mammalian cells, such as low production costs and low risk of contamination with human pathogens. Plant suspension cells, such as the Nicotiana tabacum Bright Yellow 2 (BY-2) cells, offer additional advantages, as they are easily amenable to comply with Good Manufacturing Practices (GMPs) and easily scalable, making them particularly interesting for the production of recombinant proteins of pharmaceutical value. However, issues related to these platforms, such as low yields due to degradation by endogenous proteases, have hindered their commercialisation. In this work, two recombinant protein production platforms were used, namely Nicotiana benthamiana leaves and BY-2 suspension cells. Genetic constructs encoding the RBD with a 10xHis or V5 N-terminal tag were transiently expressed in both platforms, leading to the successful production of the protein. An affinity tag was included in the genetic constructs to enable a simple and efficient downstream purification of the protein. The tag was added on the N-terminus of the protein, as previous results with a C-terminal tag had shown tag degradation. However, in this case as well, the N-terminal tag was not detected by western blots, indicating that it might have been degraded by proteases. Another construct, encoding the N-terminal domain (NTD) and the RBD of the SARS-CoV-2 S protein, namely 10xHis-NTD-RBD, was also transiently expressed in N. benthamiana leaves for the first time. Finally, the 10xHis-RBD construct was stably expressed in BY-2 cells. While the tag was not detected by western blot, the protein was successfully produced and secreted in the extracellular medium, which still makes its purification easier.