Recombinant SARS-CoV-2 RBD Protein Expression and Purification

RBD (UniProt ID P0DTC2) with Cys538Ala was expressed in SHuffle T7 E. coli cells (New England Biolabs, Ipswich, MA, USA), using a pET15b expression vector in LB medium containing 50 μg/mL ampicillin as previously reported [10 (link)]. The protein was purified by using denaturing nickel–nitrilotriacetic acid (Ni-NTA) (Wako, Tokyo, Japan) chromatography and reverse-phase (RP) high-performance liquid chromatography, lyophilized and stored at −30 °C until use [17 (link)].

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Yoshizue T., Brindha S., Wongnak R., Takemae H., Oba M., Mizutani T, & Kuroda Y. (2023). Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization. International Journal of Molecular Sciences, 24(13), 10583.

Publication 2023

SHuffle T7 E. coli cells Ni-NTA

Ampicillin Cells Chromatography E coli Nickel nitrilotriacetic acid Protein Reverse phase liquid chromatography Vector

Corresponding Organization : Tokyo University of Agriculture and Technology

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Variable analysis

independent variables

Expression of RBD (UniProt ID P0DTC2) with Cys538Ala mutation

dependent variables

Purification of the expressed protein by denaturing nickel–nitrilotriacetic acid (Ni-NTA) chromatography and reverse-phase (RP) high-performance liquid chromatography
Lyophilization and storage of the purified protein at −30 °C

control variables

SHuffle T7 E. coli cells (New England Biolabs, Ipswich, MA, USA) used for expression
PET15b expression vector used
LB medium containing 50 μg/mL ampicillin used for expression

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