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Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics
Sundarrajan, Sudarson; Rao, Sneha; Padmanabhan, Sriram.
  • Sundarrajan, Sudarson; Rangadore Memorial Hospital. Sri Shankara Research Center. Bangalore. IN
  • Rao, Sneha; Rangadore Memorial Hospital. Sri Shankara Research Center. Bangalore. IN
  • Padmanabhan, Sriram; Rangadore Memorial Hospital. Sri Shankara Research Center. Bangalore. IN
Braz. j. microbiol ; 49(4): 848-855, Oct.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-974300
ABSTRACT
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 ∼35 mg/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 10 IU/mL, which is interesting and novel.
Assuntos


Texto completo: DisponíveL Índice: LILACS (Américas) Assunto principal: Proteínas de Bactérias / Vírus da Hepatite B / Reação em Cadeia da Polimerase / Thermus thermophilus / Clonagem Molecular / Recombinases Tipo de estudo: Estudo diagnóstico Idioma: Inglês Revista: Braz. j. microbiol Assunto da revista: Microbiologia Ano de publicação: 2018 Tipo de documento: Artigo País de afiliação: Índia Instituição/País de afiliação: Rangadore Memorial Hospital/IN

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Texto completo: DisponíveL Índice: LILACS (Américas) Assunto principal: Proteínas de Bactérias / Vírus da Hepatite B / Reação em Cadeia da Polimerase / Thermus thermophilus / Clonagem Molecular / Recombinases Tipo de estudo: Estudo diagnóstico Idioma: Inglês Revista: Braz. j. microbiol Assunto da revista: Microbiologia Ano de publicação: 2018 Tipo de documento: Artigo País de afiliação: Índia Instituição/País de afiliação: Rangadore Memorial Hospital/IN