ISOLASI DAN KARAKTERISASI MUTAN, PDI1 SENSITIF TEMPERATUR RAGI SACCHAROMYCES CEREVISIAE

• Main Author: Helwati, Hira
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/4796
• Institution: Institut Teknologi Bandung

<b>Abstract:<p align=\"justify\"> <br /> Disulphide bonds between thiol groups of cysteine residues stabilize tertiary structure of secretory and membrane proteins. Disulphide bonds formation occur in the lumen endoplasmic reticulum (ER) and catalyzes by Protein Disulphida Isomerase (PDI).<p align=\"justify\"> <br /> PDI is essential for viability of Saccharomyces cereviasiae. Yeast PDI encoded by PDII gene consisting of 530 amino acid residues that contains a,b,b\', a\' and c domains. The a and a\' domains consisting of -CGHC- sequences are the active site of PDI, while b and b\' domains are putative binding site and c domain consisting -HDEL is retention signal to ER.<p align=\"justify\"> <br /> In vitro mutagenesis using hydroxylamine as mutagen in the region of b and b\' of the gene were carried out to investigate more about the amino acid responsible for the function of this domain. The mutant gene carried by pRS314 pdil plasmid was introduced on S. cerevisiae 2736 using transformation and Plasmid shuffling method.<p align=\"justify\"> <br /> Plasmid shuffling were done using YNB media containing 5 Fluoroorotic acid to select the transformans which carried only pRS314-pdi1. Further investigation on the selected trasformans resulted three temperature sensitive transformans namely YITB 245, YITB 351 and YITB 359. These temperature sensitive transformans secreted the same level killer toxin compared to the wild type. Further investigation indicated that there were no alteration in the nucleotide sequence of the HpaI-Bg1II fragment of YITB 245 and YITB 359.