Recombinant Engineering of SARS-CoV-2 Spike and N proteins

NIH Pandemic-Era Grants

Pandemic Era Grants

2020

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Principal Investigator: David  Margulies
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $38,570
Funding agency: National Institute of Allergy and Infectious Diseases

Our goal in this project is to explore published structures of the SARS-CoV-2 Spike protein with a focus on its receptor binding domain (RBD), in efforts to generate recombinant molecules of increased thermal stability. To this end we have examined published structures and bioinformatically have made a number of predictions of site-directed mutants that may yield molecules of increased stability. Initial studies have confirmed our ability to produce the wild type RBD in good yield from E. coli expression vectors, to produce the molecule in high purity through multiple chromatographic steps, and to confirm its ability to bind specific antibodies. Mutational studies are in progress. In addition, we have obtained vectors for the expression of the full-length Spike protein and its lumens domain, and expression studies in mammalian systems are underway. Also, we are reengineering the angiotensin converting enzyme-2 (ACE-2) receptor for similar expression, binding, and mutagenesis studies.

Terms: <2019 novel coronavirus><2019-nCoV><Affinity><Angiotensin Converting Enzyme><Angiotensin I-Converting Enzyme><Angiotensins><Antibodies><Binding><Bio-Informatics><Bioinformatics><CD143 Antigens><Carboxycathepsin><Complex><Computing Methodologies><Dipeptidyl Peptidase A><E coli><E. coli><Engineering><Escherichia coli><Exhibits><Fc Receptor><Genetic Alteration><Genetic Change><Genetic defect><Genetics-Mutagenesis><Goals><Kininase A><Kininase II><Length><Molecular Interaction><Mutagenesis><Mutagenesis Molecular Biology><Mutation><Nucleoproteins><Peptidyl-Dipeptidase A><Protein Engineering><Proteins><Publishing><Receptor Protein><Recombinants><Resolution><SARS-CoV-2><SARS-CoV2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Series><Severe acute respiratory syndrome coronavirus 2><Single Crystal Diffraction><Site><Structure><Surface Plasmon Resonance><System><Wuhan coronavirus><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><antibody receptor><computational methodology><computational methods><computer based method><computer methods><computing method><design><designing><expression vector><genetic protein engineering><genome mutation><improved><mutant><nanobodies><nanobody><protein design><receptor><receptor binding><receptor bound><screening><sdAb><single domain antibodies>