ISOLATION AND CLONING OF PROMOTER FORMATE DEHYDROGENASE (FDH1) GENE FROM Candida orthopsilosis

• Main Author: Wiharyanti, Risma
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34600
• Institution: Institut Teknologi Bandung

Nowadays, commonly used the hepatitis B vaccine is a recombinant vaccine, which is produced through antigen expression in the methylothropic yeast. Its expression system is regulated at the level of transcription using methanol-induced promoters. Since, such technology is patented, an alternative is required to gradually minimize the patented components through exploration of local resources. This study was aimed to obtain a local yeast candidate from ITB culture collection and a methanol-induced promoter fragment from the selected yeast to be cloned in the pJET1.2 vector for fragment analysis. The promoter would be expected to substitute patented material. Five species of yeasts were screened to determine the species that could grow optimally in medium containing methanol. The tests were conducted on Candida albicans, Candida tropicalis, Candida orthopsilosis, Pichia pastoris and Saccharomyces cerevisiae. The result showed that C. orthopsilosis had the most optimum growth among the other Candida species. One of the key enzymes of methanol metabolism pathways in yeast is alcohol oxidase (AOX). However, analysis showed that there was no homologous sequence to AOX could be obtained from C. orthopsilosis genome, it may lead to another mechanism resulting in optimal growth of C. orthopsilosis in medium containing methanol. Formate dehydrogenase (FDH) is one of enzymes that is highly increased when various methylothropic yeasts were cultured in medium containing methanol in addition to AOX. High level expression of FDH1 gene is also known to be induced by methanol, therefore PFDH1 of C. orthopsilosis is a potential alternative promoter that can substitute current promoter. PFDH1 fragment was amplified by Polymerase Chain Reaction (PCR) and subsequently inserted into pJET1.2 cloning vector. The results showed that 2178 bp PFDH1 fragment was amplified and recombinant clones containing PFDH1 was obtained. Alignment of DNA sequence using BLAST showed that PFDH1 fragment was highly homologous (94%) to upstream region of FDH1 gene from C.orthopsilosis Co 90-125. Sequence analysis revealed two putative regions as upstream activating sequence (UAS). Putative UAS regions were located -1069 to -1059 and -560 to -574 bp from the start codon. Transcription factor prediction using bioinformatic program TRANSFAC showed one putative motif for Adr1p binding site located in -1653 to -1656 bp from the start codon. Further developments of the promoter to be an alternative in protein expression systems to produce antigens for vaccine that is patent-free are encouraged.