tetano
Editor, Senior Moderator
ACS Synth Biol
. 2021 Feb 3.
doi: 10.1021/acssynbio.0c00566. Online ahead of print.
Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness
Laura Jo Sherwood[SUP] 1 [/SUP], Andrew Hayhurst[SUP] 1 [/SUP]
Affiliations
Abstract
Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC[SUB]50[/SUB] in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.
Keywords: SARS-CoV-2; coronavirus; emerging pathogen; fluorescent protein; nanobody; nucleocapsid.
. 2021 Feb 3.
doi: 10.1021/acssynbio.0c00566. Online ahead of print.
Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness
Laura Jo Sherwood[SUP] 1 [/SUP], Andrew Hayhurst[SUP] 1 [/SUP]
Affiliations
- PMID: 33534552
- DOI: 10.1021/acssynbio.0c00566
Abstract
Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC[SUB]50[/SUB] in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.
Keywords: SARS-CoV-2; coronavirus; emerging pathogen; fluorescent protein; nanobody; nucleocapsid.