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Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics.

Authors
  • Sundarrajan, Sudarson1
  • Rao, Sneha1
  • Padmanabhan, Sriram2
  • 1 Rangadore Memorial Hospital, Sri Shankara Research Center, Cancyte Technologies Pvt. Ltd, Shankarapuram, Bangalore, India. , (India)
  • 2 Rangadore Memorial Hospital, Sri Shankara Research Center, Cancyte Technologies Pvt. Ltd, Shankarapuram, Bangalore, India. Electronic address: [email protected] , (India)
Type
Published Article
Journal
Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]
Publication Date
Apr 12, 2018
Identifiers
DOI: 10.1016/j.bjm.2018.03.007
PMID: 29691193
Source
Medline
Keywords
License
Unknown

Abstract

We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10IU/mL, which is interesting and novel.

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