水稻（Oryza sativa L.）两种NADP-苹果酸酶在大肠杆菌中的表达、纯化及特性分析
Expression, purification, and characterization of two NADP-malic enzymes of rice (Oryza sativa L.) in Escherichia coli.
作者信息
Cheng Yuxiang, Takano Tetsuo, Zhang Xinxin, Yu Song, Liu Dali, Liu Shenkui
机构信息
Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China.
出版信息
Protein Expr Purif. 2006 Jan;45(1):200-5. doi: 10.1016/j.pep.2005.09.018. Epub 2005 Oct 24.
NADP-malic enzymes (NADP-ME) are isozymes in plants. To clarify the diversity and function of NADP-ME isozymes in rice, we produced two active GST-fused NADP-ME proteins, NADP-ME2 and NADP-ME3 in Escherichia coli, and the fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column. After enzymatic cleavage of the GST tag, final yields were 1.4 mg/g wet cell weight (wcw) for NADP-ME2 and 3.5 mg/g wcw for NADP-ME3, respectively, and the molecular weights of NADP-ME2 and NADP-ME3 were about 65 and 62 kDa, respectively. The optimum pH is 7.3 for NADP-ME2 and 7.7 for NADP-ME3. The Km values for malate of NADP-ME2 and NADP-ME3 were 2.6 and 3.1 mM, whereas the Km values for NADP were 79 and 93 microM, respectively. The Kcat values of NADP-ME2 and NADP-ME3 for malate were about 91.7 and 96.7 s-1, respectively, and the Kcat values for NADP about 88.3 and 98.3 s-1, respectively. These results suggest that the two rice isozymes of NADP-ME in vitro have similar kinetic parameter.
NADP - 苹果酸酶（NADP - ME）是植物中的同工酶。为了阐明水稻中NADP - ME同工酶的多样性和功能，我们在大肠杆菌中产生了两种活性的谷胱甘肽 - S - 转移酶（GST）融合的NADP - ME蛋白，即NADP - ME2和NADP - ME3，并使用谷胱甘肽 - 琼脂糖4B柱通过亲和色谱法纯化融合蛋白。在对GST标签进行酶切后，NADP - ME2的最终产量为1.4 mg/g湿细胞重量（wcw），NADP - ME3的最终产量为3.5 mg/g wcw，NADP - ME2和NADP - ME3的分子量分别约为65 kDa和62 kDa。NADP - ME2的最适pH为7.3，NADP - ME3的最适pH为7.7。NADP - ME2和NADP - ME3对苹果酸的Km值分别为2.6和3.1 mM，而对NADP的Km值分别为79和93 μM。NADP - ME2和NADP - ME3对苹果酸的Kcat值分别约为91.7和96.7 s-1，对NADP的Kcat值分别约为88.3和98.3 s-1。这些结果表明，水稻中的两种NADP - ME同工酶在体外具有相似的动力学参数。
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