Optimization of Chemically Structural Refolding of Spike 1 Recombinant Protein from SARS-CoV-2 Alberta Michelle Widyadi, Fernita Puspasari, Dessy Natalia, Ihsanawati
a) Chemistry, Faculty of Mathematics and Natural Science, Bandung Institute of Technology. Jalan Ganesha 10, Bandung 40132, Indonesia.
Abstract
S1 is a subunit of spike protein from SARS-CoV-2. This subunit plays a critical role for receptor interaction with human ACE-2 and virus fusion membrane process. S1 recombinant protein expression in E. coli produce an inclusion body that indicates the lost of protein native structure. This study aims to determine the optimum additives in buffer to help stabilize the S1 refolded recombinant protein. To achieve this goal, first the competent cell of E. coli BL21 CodonPlus (DE3) RIPL is transformed with recombinant plasmid pET-16b that carries the S1 encoded gen. Then, the transformed cell is grown in LB media containing ampicillin and induced by IPTG to expressed the S1 recombinant protein. After that, the protein is isolated and solubilized using 7.3 M urea. The solubilized protein is purified with Ni-NTA affinity chromatography. Next, the purified protein is refolded with slow dilution method using three additives that were added to the refolding buffer, N-lauroylsarcosine, arginine, and sucrose. The refolded protein is also characterized with Circular Dichroism spectroscopy. A band with the size of ~75 kDa is observed in the SDS-PAGE electrophoregram lysis cell crude extract. This band correlates to size of the S1 recombinant protein from the amino acid composition. The same band size was observed in theelution part of SDS-PAGE electrophoregram purification process. Highest recovery percentage was obtained using sucrose as an additives in buffer with the number of 95,4%. Sucrose provides a preferential hydration mechanism for protein stabilization, thus protein is surrounded by more water. Circular dichroism spectra demonstrated that the refolding process have restore the structure of S1 recombinant protein to the native state.
Keywords: inclusion bodies, refolding, SARS-CoV-2 virus, spike 1 protein
