Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli.
Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).
Yu
Song
S
Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China.
Xia
Dexi
D
Luo
Qiuxiang
Q
Cheng
Yuxiang
Y
Takano
Tetsuo
T
Liu
Shenkui
S
D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010940:Plant Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D002256:Carbonic Anhydrases