Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics
Braz. j. microbiol
; 49(4): 848-855, Oct.-Dec. 2018. tab, graf
Article
in En
| LILACS
| ID: biblio-974300
Responsible library:
BR1.1
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.
Key words
Full text:
1
Index:
LILACS
Main subject:
Bacterial Proteins
/
Hepatitis B virus
/
Polymerase Chain Reaction
/
Thermus thermophilus
/
Cloning, Molecular
/
Recombinases
Type of study:
Diagnostic_studies
Language:
En
Journal:
Braz. j. microbiol
Journal subject:
MICROBIOLOGIA
Year:
2018
Type:
Article