KONSTRUKSI DAN KARAKTERISASI MUTAN PROTEIN DISULFIDA ISOMERASE RAGI YANG TIDAK MENGANDUNG DOMAIN B'

• Main Author: PURKAN
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/2198
• Institution: Institut Teknologi Bandung

Protein disulfide isomerase (PDI, EC 5.3.4.1) encoded by Saccharomyces cerevisiae PDII gene is a catalyst in oxidation of thiol groups, reduction and isomerization of disulfide bonds in protein molecules. The enzyme consist of two subunits. Each subunit contains a, b_ b', a' and c domains. The aims of this research were to construct and to characterize pdi mutant without the b' domain (pdi- Ab'). The pdi-Ab' mutant was constructed by deletion of DNA fragment corresponding to b' domain from PDI1 gene by using PCR. Deletion of DNA fragment of b' domain of PDI was undertaken by three rounds of PCR. The first round of PCR was performed to amplify the DNA fragment of a'-c domain. The 3' end of the resulted DNA fragment (± 447 bp) also contained several nucleotides complemented to the 5' end nucletide of b domain. The ± 447 bp DNA fragment was used as a primer in combination with another primer to generate a ± 1 190 by DNA fragment corresponding to a-ba'- c domain in second round PCR. The third round of PCR was performed to amplify the ± 1190 bp DNA fragment obtained from the second round PCR. The resulted DNA fragment was first subcloned into pGem T plasmid. The pGem 'I'- pdi l -Ab' recombinant was digested with Ndel / BamH1 restriction enzymes. The Ndel / BamHI ± 1190 by DNA fragment was subcloned into an E. coil pT 7.7 expression vector. The sequence of the ± 1190 hp was confirmed by dideoxy sequence analysis and found to be free of mutation. The pdil-Ab' was expressed as a protein with molecular weight of ± 45 kDa, while the molecular weight of the wild type PDI was t 60 kDa. The pdi-Ab' had 52 reduction activity of the wild type 1'Dl. This result indicates that deletion of b' doinoin reduces the cooperativity amongst other domains to bind the substrate and hence the b' domain might plays an important role in the substrate binding. The pdi-Ab' mutant appeared to be more sensitive to proteinase K. This phenomena suggest that the pdi-Ab' mutant might have different conformation stability than the wild type PDI .