Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli.
The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His(10)-ADC (77.3 kDa) was first purified by Ni-NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His(10) tag removed recombinant ADC (74.1 kDa)'s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6-9) and temperature (20-80 degrees C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.
Song
Jiaping
J
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 200433, Shanghai, China.
Zhou
Chuanwen
C
Liu
Rui
R
Wu
Xudong
X
Wu
Di
D
Hu
Xiaojian
X
Ding
Yu
Y
D002262:Carboxy-Lyases / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D003470:Culture Media; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D002262:Carboxy-Lyases; C019722:arginine decarboxylase