Expression of the E1 Protein from Chikungunya Virus in Pichia pastoris

• Main Author: Artsani Hanif Anka, Annisa
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32283
• Institution: Institut Teknologi Bandung

Chikungunya is an infection disease caused by chikungunya virus (CHIKV), which has been reemerged in Indonesia even in the world. CHIKV genom has two open reading frames (ORF) encoding four non-structural proteins (nsP1, nsP2, nsP3, nsP4) and five structural proteins (capsid, E3, 6K, E1, E2). The E1 envelope protein, one of the structural proteins has high sensitivity and selectivity to detect anti-CHIKV IgM antibodies hence it can be used as a basis of chikungunya diagnostic kit. The objective of this research was to express E1 protein from CHIKV in Pichia pastoris. The E1 gene which had been obtained from the previous research was inserted in pPICZ?A plasmid as an expression vector. Pichia pastoris was transformed with the pPICZ?A plasmid using electroporation and resulting some transformants. Colony PCR using the AOX1 primers confirmed nine positive transformants containing the E1 gene. Screening on media with high concentration of zeocin showed that all transformants had the multicopy of the E1 gene. The E1 protein was expressed in Pichia pastoris KM71 and Pichia pastoris GS115 as the expression host. Based on Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoregram, there was a band with a molecular weight of 57 kDa corresponding to the E1 protein. A P. pastoris- pPICZ?A-E1 GS115 transformants displayed a thick SDS-PAGE band of E1 on the fifth day of induction with 2% (v/v) methanol. The E1 protein was purified by Ni-NTA affinity chromatography. The SDS-PAGE electrophoregram showed that there is no 57 kDa band on the elution lane. It showed that purifiying of the E1 protein should be done using different chromatographic methods.