OPTIMIZATION OF CLONING CONDITIONS FOR OVERPRODUCTION OF RECOMBINANT L1 PROTEIN IN HANSENULA POLYMORPHA AND PICHIA PASTORIS

• Main Author: Wilianto, Handi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66323
• Institution: Institut Teknologi Bandung

Development of a Virus-like particle (VLP) subunit vaccine for prevention of a viral infection is currently in great demand because it is safer, has a good quality and effective. The recombinant L1 HPV33 protein (rL1HPV33) is the main capsid protein of Human papillomavirus 33 (HPV33) that can be spontaneously assembled into VLP. VLP which is formed from L1 HPV33 protein is the main raw material in the manufacture of prophylactic vaccines against HPV33. This study was conducted to optimize cloning conditions as a preparation for producing L1 HPV33 protein in yeast host cells Hansenula polimorpha NCYC 495 Leu1.1 and Pichia pastoris GS115. This research begins with isolation and confirmation of pHIPH4_L1HPV33 and pD902_L1HPV33 plasmids which have been successfully transformed into Escherichia coli DH5?host cells, plasmid linearization, electroporation of plasmids into yeast host cells, primer design, confirmation of yeast transformant and production of yeast total protein. Isolation and confirmation of pHIPH4_L1HPV33 and pD902_L1HPV33 plasmids have been successfully carried out by migration and restriction enzyme analysis, as well as sequencing. Optimal conditions for linearization of the pHIPH4¬_L1HPV33 plasmid is obtained by using the StuI enzyme as much as 10 units for every 1000 ng plasmid for 24 hours at 37 °C and the pD902_L1HPV33 plasmid using SacI as much as 10 units for each 1000 ng plasmid for 4 hours at 37 °C. Electroporation of pHIPH4_L1HPV33 plasmid into yeast cells Hansenula polymorpha NCYC 495 Leu.1.1 has not been successfully carried out, so it is necessary to be optimized further. Meanwhile, electroporation of pD902_ L1HPV33 plasmid into yeast host cells Pichia pastoris GS115 was successfully carried out using competent cells that have been prepared by adding 1 M sorbitol and T buffer and the electroporation conditions with a voltage of 1.5kV. The primers were successfully designed for confirming the correctness of inserted genes, plasmids, yeast host cells, integration into chromosomes and sequencing purposes. The production of total yeast protein and L1 HPV33 protein in Pichia pastoris GS115 by induction of 0.5 and 1% methanol for 96 hours at 30°C in the dark has been successfully carried out. The presence of protein has been characterized using SDS-PAGE and the optimal method for yeast cell lysis was using glass beads 15 cycles (30 seconds vortex, 30 seconds incubation in ice-bath).