Purification and characterization of a functionally active Mycobacterium tuberculosis pyrroline-5-carboxylate reductase.
Pyrroline-5-carboxylate reductase (P5CR) plays an important role in the survival of Mycobacterium tuberculosis and is related to virulence of this pathogen. RT-PCR analysis indicated that proC, encoding P5CR, was expressed at the transcriptional level cultured in vitro. The His-rMtP5CR with an N-terminal His-tag (His-rMtP5CR) was firstly purified in Escherichia coli and rMtP5CR was obtained by removal of the N-terminal fusion partner using enterokinase. His-rMtP5CR had considerable beta-pleated sheet analyzed by circular dichroism spectroscopy. The effect of pH, temperature, cations, denaturants, and detergents on the purified enzyme activity and stability was characterized. The N-terminal fusion partner was found to have very little effect on the biochemical properties of P5CR.
Yang
Yanping
Y
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China.
Xu
Shengfeng
S
Zhang
Min
M
Jin
Ruiliang
R
Zhang
Lu
L
Bao
Jialing
J
Wang
Honghai
H
D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011762:Pyrroline Carboxylate Reductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013696:Temperature
D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; D011762:Pyrroline Carboxylate Reductases; C509372:pyrroline-5-carboxylate reductase, M tuberculosis