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

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Date
2017-08
Authors
Ahsad Ahmad@Azhar Ali, Zeheera Ali
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Publisher
Universiti Sains Malaysia
Abstract
Thermostable 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.
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Keywords
Thermostable DNA polymerase enzyme I , for disease detection through nucleic acid-based diagnostics
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