RRC ID 79423
Author Tsukamoto A, Jae Man L, Oyama K, Masuda A, Mon H, Ueda T, Kusakabe T.
Title Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.
Journal Protein Expr Purif
Abstract A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.
Volume 218
Pages 106450
Published 2024-6-1
DOI 10.1016/j.pep.2024.106450
PII S1046-5928(24)00022-6
PMID 38395208
MeSH Animals Bombyx* / genetics Bombyx* / metabolism COVID-19* HEK293 Cells HeLa Cells Humans Nucleopolyhedroviruses* Protein Binding Recombinant Proteins SARS-CoV-2 / genetics SARS-CoV-2 / metabolism Spike Glycoprotein, Coronavirus / chemistry
IF 1.513
Resource
Silkworms silkworm