EKSPRESI DAN PEMURNIAN PROTEIN BERBOBOT MOLEKUL 112 KDA ISOLAT CIMANGGU DI ESCHERICHIA COLI
DNA Polymerase catalyzes the formation of phosphodiester bonds between deoxyribonucleotides and the 3'-OH end of a DNA chain. The DNA polymerase gene of the thermophilic bacteria collected from Cimanggu hot spring was cloned in plasmid pITB6 and expressed in Escherichia coli. the properties of...
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| Format: | Theses |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/2069 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | DNA Polymerase catalyzes the formation of phosphodiester bonds between deoxyribonucleotides and the 3'-OH end of a DNA chain. The DNA polymerase gene of the thermophilic bacteria collected from Cimanggu hot spring was cloned in plasmid pITB6 and expressed in Escherichia coli. the properties of recombinant DNA Polymerase havn't been discovered. This project was aimed to analyze the expression level and the purity of the protein with molekul weight 112 kDa which was assumed as recombinant DNA Polymerase. The expression level of the protein was observed by SDSPAGE and analysed by Photocap Programme. The overexpressed recombinant protein in E. coli after 4 hours tryptophan induction was about 30% of the total protein. The crude extract protein was initially purified by heat treatment at 60°C for 20 minutes, followed by ammonium sulphate fractionation and chromatography column on DEAE-cellulose. The 40-60% ammonium sulphate saturated fraction with a highest protein content which was further purified using DEAE-cellulose chromatography resulted a recombinant protein with 85% relatif homogeneity. |
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