SARS-CoV-2 Spike Protein Expression and Purification

Protein expression and purification were largely the same as described previously^{7 (link),28 (link)}. Twin-strep tagged BA.4/5 and BA.4+all spikes were transiently expressed in HEK293T cells and purified with Strep-Tactin XT resin (IBA Lifesciences). Plasmids encoding BA.4/5 and BA.4+all RBD were transiently expressed in Expi293F™ Cells (ThermoFisher), cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30 °C with 8% CO₂ for 4 days. The harvested medium was concentrated using a QuixStand benchtop system. His-tagged RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare), followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare).

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Liu C., Das R., Dijokaite-Guraliuc A., Zhou D., Mentzer A.J., Supasa P., Selvaraj M., Duyvesteyn H.M., Ritter T.G., Temperton N., Klenerman P., Dunachie S.J., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2024). Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection. Nature Communications, 15, 3284.

Publication 2024

Strep-Tactin XT resin Expi293F™ Cells FreeStyle™ 293 Expression Medium HisTrap nickel column Superdex 75 10/300 GL gel filtration column

Corresponding Organization :

Other organizations : University of Oxford, Wellcome Centre for Human Genetics, Oxford University Hospitals NHS Trust, Medway School of Pharmacy, University of Kent, Diamond Light Source

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Variable analysis

independent variables

Expression of twin-strep tagged BA.4/5 and BA.4+all spikes in HEK293T cells
Expression of plasmids encoding BA.4/5 and BA.4+all RBD in Expi293F™ Cells

dependent variables

Purification of twin-strep tagged BA.4/5 and BA.4+all spikes using Strep-Tactin XT resin
Purification of His-tagged RBDs using a 5 mL HisTrap nickel column and Superdex 75 10/300 GL gel filtration column

control variables

Transient expression in HEK293T cells
Transient expression in Expi293F™ Cells
Culture in FreeStyle™ 293 Expression Medium at 30 °C with 8% CO2 for 4 days
Concentration using a QuixStand benchtop system

positive controls

Protein expression and purification procedures described in previous studies (references 7 and 28)

negative controls

No negative controls explicitly mentioned

Annotations

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