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
Journal Title
Journal ISSN
Volume Title
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.
Description
Keywords
Thermostable DNA polymerase enzyme I , for disease detection through nucleic acid-based diagnostics