REFOLDING OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) RECEPTOR BINDING DOMAIN (RBD) PROTEIN USING FAST DILUTION TECHNIQUE

• Main Author: Lalita Izdihar, Hana
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/72773
• Institution: Institut Teknologi Bandung

The Receptor Binding Domain (RBD) of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) can recognize the human cell receptor, Angiotensin Converting Enzyme 2 (ACE2). This interaction is the initial step of virus infection in humans. Therefore, RBD is a protein target for therapy, vaccination, and studying the pathogenicity of SARS-CoV-2. Escherichia coli expression system is an ideal host to produce recombinant proteins because of its fast growth rate and ability to produce a large amount of protein. However, RBD production in E. coli results in inclusion bodies that affects the function and structure of RBD protein. Inclusion bodies are a consequence of protein misfolding and generally result in the loss of activity. The aim of this study is to refold the recombinant SARS-CoV-2 RBD protein structure using rapid dilution and characterize the RBD structure using spectrofluorometry. The first stage of the study was to transform E. coli BL21 (DE3) cells with a recombinant plasmid containing the RBD gene. The obtained transformants were induced with isopropyl ?-D-1-thiogalactopyranoside (IPTG) to induce RBD protein expression. The RBD protein in inclusion bodies was dissolved in a buffer solution containing 8 M urea and purified by Ni-NTA affinity chromatography. The pure protein was then refolded using rapid dilution and characterized by spectrofluorometry. The transformed recombinant E. coli cells were able to grow in Luria Bertani (LB) medium containing ampicillin, and produced a band of 25 kDa on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This indicates that the cell transformation and RBD production stages were successfully conducted. The expressed protein was in the form of aggregates but was soluble in 8 M urea and produced a protein band of 50 kDa on SDS-PAGE. This result indicates the dimeric form of RBD in the denatured state. The 50 kDa protein band was more clearly visible after purification. The fluorescence spectrum after refolding showed a decrease in the percentage of fluorescence emission with decreasing urea concentration in the RBD solution. The normalized spectrum showed a maximum emission wavelength shift from red shift to blue shift. This wavelength shift is indicative of RBD conformational change from denatured to refolded state.