Expression of Envelope Protein Domain III of Dengue-1 Virus in Pichia pastoris KM71

• Main Author: DWIPUTRA (NIM : 10513016), NICODEMUS
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29620
• Institution: Institut Teknologi Bandung

<p align="justify">Severe dengue is a disease caused by dengue virus infection and spread by Aedes aegypti. There were 71.688 severe dengue cases in Indonesia throughout 2014 with 641 deaths. The symptoms of the disease is often indistinctive, therefore a rapid and accurate diagnosis method is needed so that proper medical care can be performed. Envelope protein (E) is a structural protein found in the surface of the dengue virion that has a receptor binding site. Domain III of E protein which acts in receptor binding is known to reside at virion surface and possessing a number of neutralizing epitopes thus making it a suitable target for development of diagnostic kit for severe dengue detection. The purpose of this research was to construct Pichia pastoris KM71 harboring pPICZ&#945;A-EDIII-D1 and to optimize the expression of EDIII-D1 protein in P. pastoris KM71. P. pastoris KM71 was transformed using pPICZ&#945;A-EDIII-D1 carrying synthetic EDIII-D1 gene. Gene multicopy selection was carried out using YPD medium with 1000 &#956;g/mL and 2000 &#956;g/mL zeocin. The presence of EDIII-D1 gene in P. pastoris KM71 chromosome by PCR (Polymerase Chain Reaction) using AOX1 forward primer and AOX1 reverse primer showed a band at 960 bp matching the EDIII-D1 recombinant protein coding DNA fragment. EDIII-D1 protein expression was carried out using 2% methanol induction every 12 hours for 72 hours. Electrophoregram analysis of Tricine SDS-PAGE showed a 18 kDa protein band corresponding to the expected molecular mass of EDIII-D1. The optimum condition for EDIII-D1 expression was at 2% methanol induction for 72 hours.<p align="justify">