SUBCLONING AND EXPRESSION OF RBD-FOLDON ON HANSENULA POLYMORPHA

• Main Author: Griani Gusti, Ade
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64750
• Institution: Institut Teknologi Bandung

Coronavirus Disease-2019 (Covid-19) is a respiratory system disorder caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Preventive action in the form of vaccination continue to be carried out to suppress the spread of the SARS-CoV-2 virus. One of the vaccines used is protein-based SARS-CoV-2. The RBD protein is domain of Spike (S) protein, this S protein plays an important role in interacting with human ACE-2 (Angiotensin-Converting Enzyme-2) receptor cells so the virus can enter the host cell. Vaccine efficacy is influenced by the stability and ability of the antigen to trigger the body's immune response. Therefore, the RBD domain needs to be modified through the formation of fusion with the Foldon protein. Foldon protein is a domain located at the C-terminal of the fibritin protein of bacteriophage T4 which plays a role in the formation of fibritin protein trimerization. The RBD-Foldon fusion is expected to produce the RBD-Foldon trimer. In this study, yeast Hansenula polymorpha NCYC 495 leu 1.1 was used to express RBD-Foldon protein and accompanied by the addition of ?- MFS (?-Mating Factor Signal) secretion signal. The aims of this study were to construct pHIPX4-?-MFS-RBD-Foldon, construct H. polymoprha NCYC 495-?- MFS-RBD-Foldon, and to do RBD-Foldon protein expression and optimization. The ?-MFS-RBD-Foldon gene was amplified by PCR (Polymerase Chain Reaction) and pPICZ?-RBD-Foldon as a template. The amplicon was then ligated to the pGEMT clonning plasmid and produced pGEMT-?-MFS-RBD-Foldon. Then the ?-MFS-RBD-Foldon fragment that was cut with the HindIII and SalI enzymes was ligated with the expression plasmid pHIPX4 which had also been cut with the same two enzymes. The resulting recombinant plasmid was named pHIPX4-?-MFS-RBD-Foldon. The recombinant plasmid was integrated into the H. polymorpha NCYC 495 chromosome by electrotransformation method. RBD- Foldon expression was carried out on methanol-induced production media. The construction of the pGEMT-?-MFS-RBD-Foldon plasmid was successfully carried out and a plasmid of ~4 kb was obtained. The recombinant plasmid pHIPX4-?–MFS-RBD-Foldon of ~8 kb had also been successfully constructed and the nucleotide sequence obtained corresponds to the early synthetic RBD- Foldon gene. H. polymorpha NCYC 495 containing the gene encoding ?–MFS- RBD-Foldon was also obtained. The H. polymorha transformant was able to grow on YND (Yeast Nitrogen Dextrose) selective media without amino acids which indicated that the recombinant plasmid carrying the gene encoding the Sc-LEU2 gene had been integrated into the yeast chromosome. The expression of two transformant colonies with 0.5% methanol induction for 48 hours showed the presence of a protein measuring ~75 kDa which is a measure of the RBD-Foldon trimer. The RBD-Foldon protein was obtained on the media which indicated that the ?–MFS secretion signal was functioning properly. The optimum condition of RBD-Foldon expression in H. polymorpha NCYC 495 was obtained by induction of 2% methanol, OD600 cell density of 20, induction time of 72 hours, and 10% aeration in BMMY medium.