Cloning and Expression of CR3022 and ACE2

The variable regions of the heavy and light chains of CR3022^{17 (link)} were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. Likewise, ACE2 (residues 1–615) was cloned into TGEX-HC. The DNA was then transfected into Expi293F cells (Thermo Fisher Scientific) by using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) following the provided protocol, and the cells were incubated in a humidified incubator at 37 °C and 8% CO₂ for 5 days. The cells were then centrifuged at 5500 × g for 20 min. The supernatant media was diluted twofold in PBS and run through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA start (Cytiva) and controlled by Unicorn start 1.0 software (Cytiva) according to the manufacturer’s operation manual to purify the proteins. CR3022 and ACE2-Fc were quantified by using the BCA assay (Thermo Scientific).

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Chiba S., Frey S.J., Halfmann P.J., Kuroda M., Maemura T., Yang J.E., Wright E.R., Kawaoka Y, & Kane R.S. (2021). Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization. Communications Biology, 4, 597.

Publication 2021

Expi293F cells ExpiFectamine Transfection Kit MabSelect SuRe column ÄKTA start Unicorn start 1.0 software BCA assay

Ace2 Antibody Assay Cells Cr3022 Light Proteins Transfection Vectors

Corresponding Organization :

Other organizations : University of Wisconsin–Madison, Georgia Institute of Technology

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Variable analysis

independent variables

Cloning of the variable regions of the heavy and light chains of CR3022 into the TGEX-HC and TGEX-LC vectors, respectively
Cloning of ACE2 (residues 1–615) into TGEX-HC
Transfection of the DNA into Expi293F cells using the ExpiFectamine Transfection Kit

dependent variables

Purification of CR3022 and ACE2-Fc proteins using the MabSelect SuRe column
Quantification of CR3022 and ACE2-Fc using the BCA assay

control variables

Incubation of the transfected Expi293F cells in a humidified incubator at 37 °C and 8% CO2 for 5 days
Centrifugation of the cells at 5500 × g for 20 min

controls

No positive or negative controls were explicitly mentioned in the provided information.

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