KONSTRUKSI DAN EKSPRESI TINGGI GEN DNA POLIMERASE BAKTERI TERMOFILIK STRAIN LOKAL (CIMANGGU) DI ESCHERICHIA COLI

• Main Author: YOHANDINI KUSUMAWATI, HENI
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/2088
• Institution: Institut Teknologi Bandung

Recently, research on thermophilic bacteria are extensively carried out. The thermostable enzyme produced by the thermophilic bacteria could be used either for industrial application or basic sciences development. Among the commercially used thermostable enzyme is DNA polymerase, the enzyme used in PCR process. Some of the commercially available DNA polymerase have some advantage and disadvantage properties. DNA polymerase which has unique properties are still need to be explored. A few thermophilic bacterias were isolated from hot spring around Bandung city, West Java. DNA polymerase gene from one of these bacterias was cloned and sequenced. The aim of this research was emphasized on the expression of the gene. pMAL-c2 plasmid was used to express the gene in E. coll. A new restriction site was constructed on the 5' and 3' coding region of the gene to make it compatible with the vector. In addition, pMAL-c2 was modified to simplify the ligation system. The recombinant plasmid which carry the DNA polymerase-malE fusion gene was obtained and was confirmed by restriction analysis. The recombinant plasmid was used to express DNA polymerase in E. coll. SDS-PAGE of the crude extract showed that the fusion protein was expressed. However, the polymerase activity of the crude extract still indicated an unsatisfied result.