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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Keywords
Production Of Uvrd Helicase And Mutl Recombinant Proteins , For Helicasedependent Isothermal Amplificatio