SARS-CoV-2 Spike Protein Biotinylation

SARS CoV-2 Spike trimer (S-2P) and subdomains (NTD, RBD-SD1, S1) were produced by transient transfection of 293 Freestyle cells as previously described (4 (link)). Avi-tagged S1 was biotinylated using the BirA biotin-protein ligase reaction kit (Avidity, #BirA500) according to the manufacturer’s instructions. The S-2P, RBD-SD1, and NTD proteins were produced by an in-column biotinylation method as previously described (5 (link)). Successful biotinylation was confirmed using Bio-Layer Interferometry, by testing the ability of biotinylated protein to bind to streptavidin sensors. Retention of antigenicity was confirmed by testing biotinylated proteins against a panel of cross-reactive SARS-CoV and SARS CoV-2 human monoclonal antibodies. Biotinylated probes were conjugated using either allophycocyanin (APC)-, Ax647-, BV421-, BV786-, BV711-, or BV570-labeled streptavidin. Reactions were prepared at a 4:1 molecular ratio of biotinylated protein to streptavidin, with every monomer labeled. Labeled streptavidin was added in ⅕ increments and in the dark at 4°C (rotating) for 20 min in between each addition. Optimal titers were determined using splenocytes from immunized mice and validated with SARS CoV-2 convalescent human PBMC.

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Wang L., Zhou T., Zhang Y., Yang E.S., Schramm C.A., Shi W., Pegu A., Oloniniyi O.K., Henry A.R., Darko S., Narpala S.R., Hatcher C., Martinez D.R., Tsybovsky Y., Phung E., Abiona O.M., Antia A., Cale E.M., Chang L.A., Choe M., Corbett K.S., Davis R.L., DiPiazza A.T., Gordon I.J., Hait S.H., Hermanus T., Kgagudi P., Laboune F., Leung K., Liu T., Mason R.D., Nazzari A.F., Novik L., O’Connell S., O’Dell S., Olia A.S., Schmidt S.D., Stephens T., Stringham C.D., Talana C.A., Teng I.T., Wagner D.A., Widge A.T., Zhang B., Roederer M., Ledgerwood J.E., Ruckwardt T.J., Gaudinski M.R., Moore P.L., Doria-Rose N.A., Baric R.S., Graham B.S., McDermott A.B., Douek D.C., Kwong P.D., Mascola J.R., Sullivan N.J, & Misasi J. (2021). Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants. Science (New York, N.y.), 373(6556), eabh1766.

Publication 2021

BirA biotin-protein ligase reaction kit

Allophycocyanin Antigenicity Biotin ligase Biotinylation Cells Cross reactive Human Interferometry Mice Monoclonal antibodies Protein Retention Sars cov Sars cov 2 Streptavidin Transfection Transient

Corresponding Organization : National Institutes of Health

Other organizations : University of North Carolina at Chapel Hill, Frederick National Laboratory for Cancer Research, National Health Laboratory Service, University of the Witwatersrand, South African Medical Research Council

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Variable analysis

independent variables

Transient transfection of 293 Freestyle cells to produce SARS CoV-2 Spike trimer (S-2P) and subdomains (NTD, RBD-SD1, S1)
Biotinylation methods: Avi-tagged S1 using BirA biotin-protein ligase reaction kit, and S-2P, RBD-SD1, and NTD using in-column biotinylation
Conjugation of biotinylated probes with different fluorescent streptavidin labels (APC, Ax647, BV421, BV786, BV711, BV570)

dependent variables

Successful biotinylation confirmed by Bio-Layer Interferometry
Retention of antigenicity confirmed by testing biotinylated proteins against a panel of cross-reactive SARS-CoV and SARS CoV-2 human monoclonal antibodies
Optimal titers determined using splenocytes from immunized mice and validated with SARS CoV-2 convalescent human PBMC

control variables

Molecular ratio of biotinylated protein to streptavidin kept at 4:1 with every monomer labeled
Labeling reactions prepared in the dark at 4°C with 20 min incubation between each addition of streptavidin

positive controls

Splenocytes from immunized mice
SARS CoV-2 convalescent human PBMC

negative controls

Not explicitly mentioned

Annotations

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