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10.1021/acssynbio.0c00566 http://scihub22266oqcxt.onion/10.1021/acssynbio.0c00566 33534552!7875338!33534552     free     free     free Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 278.79999999999995 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 278.79999999999995 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       ACS+Synth+Biol 2021 ; 10 (2): 379-390 Nephropedia Template TP {{tp|p=33534552|t=2021. Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness |pdf=|usr=}}{{33534552}} gab.com Text Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness JO: ACS Synth Biol http://www.kidney.de/pget.php?&tw=1&pmid=33534552 #FOAvip 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(50) 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. Twit Text FOAVip Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness ????ACS Synth Biol http://www.kidney.de/pget.php?&tw=1&pmid=33534552 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=33534552 #FOAvip English Wikipedia <ref>{{Cite pmid|33534552}}</ref> Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness #MMPMID33534552 Sherwood LJ; Hayhurst A ACS Synth Biol 2021[Feb]; 10 (2): 379-390 PMID33534552show ga 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(50) 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. |*Pandemics/prevention & control[MESH] |Antibodies, Viral/genetics[MESH] |Antibody Specificity/genetics[MESH] |COVID-19/*epidemiology/immunology/*virology[MESH] |Communicable Diseases, Emerging/virology[MESH] |Coronavirus Nucleocapsid Proteins/*genetics/immunology[MESH] |Escherichia coli/genetics[MESH] |Fluorescent Antibody Technique[MESH] |Genes, Synthetic[MESH] |Genes, Viral[MESH] |HEK293 Cells[MESH] |Humans[MESH] |Molecular Probes/*genetics/immunology[MESH] |Peptide Library[MESH] |Phosphoproteins/genetics/immunology[MESH] |Recombinant Fusion Proteins/genetics/immunology[MESH] |SARS-CoV-2/*genetics/*immunology/pathogenicity[MESH] |Single-Domain Antibodies/genetics[MESH]Toolkit for Quickly Generating and Characterizing Molecular Probes Spe(epmc).pdf DeepDyve Pubget Overpricing