SARS-CoV-2 Spike Protein Variant Expression

The S constructs contained the following mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3): deletion (Δ) of H69, V70 and Y144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H in B.1.1.7; L18F, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, D614G, and A701V in B.1.351; and L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I in P.1. They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Pst I/Not I in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (Thermo Fisher Scientific). All S constructs were verified by Sanger sequencing and subsequently produced in human embryonic kidney (HEK) 293F cells (Thermo Fisher Scientific) and purified as previously described (25 (link)). The RBD constructs contained the following mutations: N501Y in B.1.1.7; K417N, E484K, and N501Y in B.1.351; K417T, E484K, and N501Y in P.1; and E484K as a single mutant. They were made by introducing the mutations into the SARS-CoV-2 RBD (331 to 528 amino acids)–StrepII construct provided by D. Lakshamanane (42 (link)) using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All RBD constructs were produced in ExpiCHO cells and purified as previously described (43 ).

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Caniels T.G., Bontjer I., van der Straten K., Poniman M., Burger J.A., Appelman B., Lavell H.A., Oomen M., Godeke G.J., Valle C., Mögling R., van Willigen H.D., Wynberg E., Schinkel M., van Vught L.A., Guerra D., Snitselaar J.L., Chaturbhuj D.N., Cuella Martin I., Moore J.P., de Jong M.D., Reusken C., Sikkens J.J., Bomers M.K., de Bree G.J., van Gils M.J., Eggink D, & Sanders R.W. (2021). Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination. Science Advances, 7(36), eabj5365.

Publication 2021

gBlock gene fragments Gibson Assembly HEK) 293F cells QuikChange Site-Directed Mutagenesis Kit

Amino acids Cells Deletion Embryonic Hexahistidine Human Kidney Mutations Sars cov 2 Site directed mutagenesis Vector

Corresponding Organization : Cornell University

Other organizations : Vrije Universiteit Amsterdam, Public Health Service of Amsterdam

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SARS-CoV-2 Variant S-Protein Antigen Constructs

The S-protein antigen constructs representing the different VOCs contained the following mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3): deletion (Δ) of H69, V70 and Y144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H in Alpha (B.1.1.7); L18F, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, D614G, and A701V in Beta (B.1.351); and L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I in Gamma(P.1). The genes were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Pst I/Not I in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (Thermo Fisher Scientific). All S constructs were verified by Sanger sequencing and the protein was subsequently produced in human embryonic kidney (HEK) 293 F cells (Thermo Fisher Scientific) and purified as previously described^{4 (link),9 (link)}.

van Rijswijck D.M., Bondt A., Hoek M., van der Straten K., Caniels T.G., Poniman M., Eggink D., Reusken C., de Bree G.J., Sanders R.W., van Gils M.J, & Heck A.J. (2022). Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern. Nature Communications, 13, 6103.

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SARS-CoV-2 Spike Protein Variant Expression

The mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3) in the S proteins are depicted in S1 Table. The S constructs were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (ThermoFisher) [32 (link)]. All S constructs were verified by Sanger sequencing, subsequently produced in HEK293F cells (ThermoFisher), and purified as previously described [32 (link)].

van Gils M.J., Lavell A., van der Straten K., Appelman B., Bontjer I., Poniman M., Burger J.A., Oomen M., Bouhuijs J.H., van Vught L.A., Slim M.A., Schinkel M., Wynberg E., van Willigen H.D., Grobben M., Tejjani K., van Rijswijk J., Snitselaar J.L., Caniels T.G., Vlaar A.P., Prins M., de Jong M.D., de Bree G.J., Sikkens J.J., Bomers M.K, & Sanders R.W. (2022). Antibody responses against SARS-CoV-2 variants induced by four different SARS-CoV-2 vaccines in health care workers in the Netherlands: A prospective cohort study. PLoS Medicine, 19(5), e1003991.

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Structural Analysis of SARS-CoV-2 Spike Variants

The 2P-stabilized S proteins
of the Wuhan strain (WT) and B.1.351
variant were described previously.^{12 (link),55 (link)} The B.1.351
construct contained the following mutations compared to the WT variant
(Wuhan Hu-1; GenBank: MN908947.3): L18F, D80A, D215G, L242H, R246I,
K417N, E484K, N501Y, D614G, and A701V. Both S constructs were produced
in HEK293F suspension cells (ThermoFisher) and purified as previously
described.^{12 (link)} For the human ACE2 receptor,
soluble ACE2 was generated as described previously^{12 (link)} by using a gene encoding amino acids 18–740 of ACE2.
The IgGs and Fab fragments used in this study were produced as previously
described.^{12 (link),26 (link)}

Yin V., Lai S.H., Caniels T.G., Brouwer P.J., Brinkkemper M., Aldon Y., Liu H., Yuan M., Wilson I.A., Sanders R.W., van Gils M.J, & Heck A.J. (2021). Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses. ACS Central Science, 7(11), 1863-1873.

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Variable analysis

independent variables

Mutations in the SARS-CoV-2 Spike (S) protein constructs compared to the Wuhan Hu-1 (WT) variant

dependent variables

Spike protein expression and purification in HEK 293F and ExpiCHO cells

control variables

Pst I/Not I expression vector containing a hexahistidine (his) tag
Sanger sequencing to verify S constructs
Previously described protocols for protein production and purification (25 and 43)

positive controls

SARS-CoV-2 RBD (331 to 528 amino acids)–StrepII construct provided by D. Lakshamanane (42)

negative controls

Not explicitly mentioned

Annotations

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