Purification of Recombinant Nucleocapsid Protein of SARS-CoV-2 Yasmin Rizkya Putri, Ihsanawati
Institut Teknologi Bandung
Abstract
Nucleocapsid protein or N protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the main antigen recognized by the human immune system. Therefore, N protein is an important target for developing diagnostic tests and anti-SARS-CoV-2 vaccines. The aim of this study was to produce and purify recombinant N protein of SARS-CoV-2. The research begun by transforming E. coli BL21 CodonPlus (DE3) RIPL cells with a recombinant plasmid containing the SARS-CoV-2 N coding gene so that these cells could grow in the antibiotic ampicillin medium. Successful transformation and production of protein N were confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The crude protein extract then subjected to purification via affinity chromatography and cation exchange. Purification of protein N using Ni-NTA affinity resin involves the coordination of histidine residues in protein N with Ni2+ ions present in the resin, forming covalent bonds. Protein N can be eluted from the resin using a buffer solution containing imidazole. For additional purification, cation exchange chromatography was employed, capitalizing on the isoelectric point (pI) of protein N, which is 10.07. By utilizing a Tris-HCl buffer with a pH of 8.0, protein N becomes positively charged. The electrostatic interactions between the functional groups on the resin and the protein can be disrupted by buffer solutions containing different concentrations of NaCl. The SDS-PAGE analysis of the crude protein N extract displayed a prominent band at approximately 45 kDa. However, purification using Ni-NTA resin resulted in closely spaced bands around 45 kDa, indicating the presence of protein impurities. Subsequent purification using HiTrap Capto S cation exchange resin revealed a single chromatogram peak with a concentration of 30 mAU, indicating the purity of protein N. The purified SARS-CoV-2 protein N can be utilized for future development of diagnostic kits.
Keywords: COVID-19, SARS-CoV-2, NCoV2, E. coli BL21 CodonPlus (DE3) RIPL
