Production Of Uvrd Helicase And Mutl Recombinant Proteins For Helicasedependent Isothermal Amplification

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Date
2015-10
Authors
SEPULOH MANIAM, SANGGETHA PERIYA
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Abstract
Helicase-dependent amplification (HDA) method is an alternative gene amplification method besides PCR. This method uses a DNA helicase to unwind double-stranded DNA into two single strands. HDA reaction can be performed at one constant temperature, eliminating the need to use a thermal cycler. In this study, two mesophilic UvrD helicases from Escherichia coli and Proteus mirabilis were produced for HDA reaction to be performed at standard room temperature. E. coli uvrD helicase gene was produced synthetically through assembly PCR method. A total of 120 oligonucleotides were designed based on E. coli strain K-12 gene sequence obtained from National Center for Biotechnology Information, USA. The oligonucleotides were divided into three parts and assembled separately. Assembled products were used as templates and amplified into three separate DNA fragments known as block 1, block 2 and block 3, using outermost oligonucleotides of each block as primers. The blocks were cloned into pCRTM-Blunt cloning vector before sending for DNA sequencing analysis. Mutations in each block were corrected and the error free blocks were combined into a full-length E. coli uvrD gene and subcloned into pET28a (+) expression vector. On the other hand, P. mirabilis uvrD helicase gene was amplified out from its genomic DNA. The gene was also cloned into pCRTM-Blunt cloning vector, sequenced and subcloned into pET28a (+) expression vector. E. coli MutL protein, an accessory protein that is required for HDA reaction was also expressed from a gene that was purchased. All the three proteins were expressed through BL21 (DE3) at 25°C, 200 rpm for 16 hours without IPTG induction. IMAC purification with Nickel ions was performed to purify the Histidine-tagged proteins. Purified proteins were buffer exchanged and were detected through SDS-PAGE and western blot analysis, showing a specific band at sizes ~82 kDa for UvrD helicases and ~68 kDa for MutL protein. Further confirmation was done through MALDI-TOF/TOF analysis and the scores significantly matched the proteins. HDA reaction showed that E. coli UvrD helicase had successfully amplified a 120 bp product at 30.0oC, 37.0oC and 42.0oC while P. mirabilis UvrD helicase worked best at 37.0oC and 42.0oC. Amplification at 25.0oC was enhanced by increasing the concentration of helicases. Around 3.0 μg/mL of E. coli UvrD helicase and 10.0 μg/mL of P. mirabilis UvrD helicase were required to amplify the 120 bp product at the standard room temperature. Overall, two DNA helicases that function well in HDA reaction were successfully produced in this study.
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Production Of Uvrd Helicase And Mutl Recombinant Proteins , For Helicasedependent Isothermal Amplificatio
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