J Integr Plant Biol. ›› 2003, Vol. 45 ›› Issue (4): 460-465.
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LIU Pu, CHEN Jia, ZHANG Hui-Xia, WANG Xue-Chen
Published:
Abstract:
Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E.coli at different temperatures. The target protein was insoluble when induced at 37 ℃, while dissolvable if induced at 22 ℃ with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent. Key words: cAMP-dependent protein kinase; fusion expression; activity assay
拟南芥PKA催化亚基基因的克隆、原核表达及其催化活性的鉴定 刘 璞 陈 珈 张会霞 王学臣 (中国农业大学生物学院植物生理生化国家重点实验室,北京100094)
摘要: 蛋白质的磷酸化与脱磷酸化是生物体内存在的一种普遍的调节方式 ,几乎参与所有的生命活动过程。利用Blast2 .0分析拟南芥基因组序列发现存在一个与动物蛋白激酶cDNA同源性的序列 ,在GenBank中比较发现它与动物的依赖cAMP的蛋白激酶 (PKA)的催化亚基 (C亚基 )有相似的特征序列。提取拟南芥 (Arabidopsisthaliana (L .)Heynh .)的总RNA ,通过RT_PCR克隆得到这一cDNA片段 ,经序列测定证实它具有完整的阅读框架 ,将其克隆至pET30a原核表达载体 ,结果表明在大肠杆菌 (E .coli)BL2 1 (DE3)中该表达质粒在IPTG诱导下表达产生大量带寡聚组氨酸标记的重组蛋白 ,该蛋白在 37℃表达时主要以包含体形式存在 ,而在 2 2℃表达时主要以可溶性蛋白形式存在。经过与组氨酸结合金属螯合树脂亲和柱层析纯化后 ,得到纯化的目的蛋白 ,其纯度达到 87%以上。活性鉴定表明其具有依赖于cAMP的蛋白激酶活性 ,而加入PKA的抑制剂 (H-8)后 ,其活性显著下降。从而证实它确实是拟南芥的PKA催化亚基。Westernblot结果显示它几乎不受ABA、NaCl等逆境的诱导。
关键词: :cAMP依赖蛋白激酶;融合表达;活性测定
通讯作者。E-mail: chenja @ public.bta.net.cn。
LIU Pu, CHEN Jia, ZHANG Hui-Xia, WANG Xue-Chen. Gene Cloning and Expression of PKA Catalytic Subunit and Its Activity in Arabidopsis thaliana[J]. J Integr Plant Biol., 2003, 45(4): 460-465.
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