Abstract
To obtain the recombinant active histidinol dehydrogenase of Mycobacterium tuberculosis (Mtb) and characterize the enzymatic properties. The gene hisD was amplified by PCR from the genomic DNA of Mtb strain H37Rv and cloned into pET28a(+) to construct the recombinant expression plasmid pET-28a-HDH. The recombinant plasmid was introduced into the cells of E. coli strain BL21 (DE3). Recombinant MtHDH was expressed after IPTG induction and enzymatic and biophysical properties was assayed after purified with Ni-NTA resin. The recombinant MtHDH was purified in active state with a specific activity of 1.788 U/mg. The optimal pH and temperature of the MtHDH were 8.3 and 45°C, respectively. The relative activity was enhanced in the presence of Mn2+, Ca2+, Zn2+and Co2+. The kinetic constants were determined: Km for NAD+ was 0.9765 mmol/L and for histidinol 2.755 |J.mol/L. Circular dichroism studies indicated that the secondary structure of the recombinant protein had about 20.5% a-helix, 40.9% P-sheet, 4.2% P-turn and 34.3% random coil at 25°C. The hisD gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of histidinol dehydrogenase. This work can facilitate the discovery of novel antimicrobial agents against tuberculosis and searching for immunologic epitopes.
Original language | English (US) |
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Title of host publication | Recent Works on Microbes and Infections in China |
Subtitle of host publication | Selected from the Journal of Microbes and Infections China |
Publisher | World Scientific Publishing Co. |
Pages | 21-30 |
Number of pages | 10 |
ISBN (Electronic) | 9789812835673 |
ISBN (Print) | 9812835660, 9789812835666 |
DOIs | |
State | Published - Jan 1 2009 |
Keywords
- Cloning
- Histidinol dehydrogenase
- L-histidinol
- Mycobacterium tuberculosis
- Nadh
- Recombinant plasmid
ASJC Scopus subject areas
- General Immunology and Microbiology
- General Medicine