Preparative Scale Production Of Recombinant Anoxybacillus DNA Polymerase I In Escherichia coli

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dc.contributor.authorAhsad Ahmad@Azhar Ali, Zeheera Ali
dc.date.accessioned2018-01-15T08:04:51Z
dc.date.available2018-01-15T08:04:51Z
dc.date.issued2017-08
dc.description.abstractThermostable DNA polymerase is an important enzyme for disease detection in nucleic acid-based diagnostics. The nucleic acid amplification is a key step in DNA detection assays. The aim of this study was to produce a thermostable DNA polymerase enzyme I for disease detection through nucleic acid-based diagnostics. The gene for the enzyme was obtained from a thermophilic bacterium known as Anoxybacillus sp. DR04. The gene encoding DNA polymerase I from Anoxybacillus sp. DR04 was cloned into Escherichia coli expression system and sequenced. It shows 99% gene identity to homologous gene in Anoxybacillus gonensis. The expression of N-terminal His6-tagged Anoxybacillus DNA polymerase was performed in E. coli Rosetta (DE3). The molecular weight of the protein was approximately 99 kDa and the yield of the target protein reached approximately 17.74 mg/mL after performing protein expression under optimized conditions. The recombinant Anoxybacillus DNA polymerase was purified on Ni2+ - NTA through immobilized metal affinity chromatograph (IMAC), and the enzyme activity was investigated. Its specific activity was 185.23 U/mg. The enzyme was able to synthesize approximately 3286 DNA strands per minute and exhibited excellent thermostability at 65°C. The recombinant Anoxybacillus DNA polymerase enzyme produced in this study is a good candidate for DNA amplifications at a single temperature of 65°C.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/5391
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectThermostable DNA polymerase enzyme Ien_US
dc.subjectfor disease detection through nucleic acid-based diagnosticsen_US
dc.titlePreparative Scale Production Of Recombinant Anoxybacillus DNA Polymerase I In Escherichia colien_US
dc.typeThesisen_US
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