EXPRESSION AND PURIFICATION OF A GENE ENCODING CHITOSANASE – RBD IN PICHIA PASTORIS X-33

• Main Author: Sukma Faradilla, Syahesti
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/77412
• Institution: Institut Teknologi Bandung

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected the respiratory tract of millions of people and caused a global pandemic for several years. SARS-CoV-2 enters host cells with receptor-binding domain (RBD) which binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells. RBD-based vaccines can stimulate a strong neutralizing antibody response, thus RBD can be the candidate for SARS-CoV-2 vaccine. Production of recombinant RBD (rRBD) was carried out using Pichia pastoris cell and Escherichia coli cell systems. rRBD in P. pastoris is soluble in specific buffers, while E. coli produces rRBD as protein precipitate. Previous studies have shown that the gene coding for chitosanase (Csn1) is highly expressed in P. pastoris. Therefore, the aim of this study was to express the RBD coding gene combined to the Csn1 gene in P. pastoris X-33. Combined expression of the Csn1-RBD gene was carried out in expression medium buffered methanolcomplex medium (BMMY) and buffered glycerol-complex medium (BMGY), by varying the production time of 3-5 days and methanol concentration of 1-3% (v/v) as inducer. The resulting Chitosanase-RBD recombinant protein was purified by affinity chromatography using Ni-NTA agarose resin. Chitosanase-RBD in the supernatant of P. pastoris X-33 has a molecular weight of ~ 59 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The size of this protein is larger than the theoretical size, which is 53.7 kDa. The results of purification with Ni-NTA resin also showed a band measuring ~ 59 kDa, but there were other bands on the SDS-PAGE electropherogram. These results indicate that the Chitosanase-RBD is partially pure. Based on chitosanase activity, recombinant protein from colonies 23 and 31 had a specific activity of 1.375 U/mg and 1.701 U/mg at 1% (v/v) methanol concentration and optimum production time was on day 4. Purification and concentration also showed an increase in purity of 5-14 times. The results of this study are expected to increase rRBD production so that it can be applied to various applications.