| 重组I型cAMP依赖的蛋白激酶调节亚基的表达,纯化和鉴定 |
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| 引用本文： | 刘旭磊,赵简.重组I型cAMP依赖的蛋白激酶调节亚基的表达,纯化和鉴定[J].生物化学与生物物理学报,1999,31(4):459-462. |
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| 作者姓名： | 刘旭磊 赵简 |
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| 作者单位： | 上海医科大学神经生物国家重点实验室!上海200032(刘旭磊，李惠明，马兰)，中国科学院上海细胞生物学研究所!上海200031(赵简，裴钢) |
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| 基金项目： | 国家自然科学基金，卫生部科学研究基金，上海市教委曙光计划基金 |
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| 摘 要： | Ｉ型蛋白激酶的调节亚基（ＲＩ）具有两个ｃＡＭＰ结合位点，对ｃＡＭＰ具有很高亲和力和特异性，我们从人神经细胞中克隆人ＲＩ亚基ｃＤＮＡ片段（编码氨基酸残基９３－３８１）并将其亚克隆至ｐＥＴ３０ａ原核表达载体，实验表明该表达质粒在大肠杆菌ＢＬ２１中，在ＩＰＴＧ诱导下，表达产生大量带聚组氨酸标记的重组ＲＩ亚基。这些蛋白质以可溶性蛋白形式存在，经组氨酸结合金属螯合树脂亲和柱层析纯化后，每０．１升培养菌可制备
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| 关 键 词： | cAMP 表达 蛋白激酶 鉴定 细胞 信号传递 |
Expression, Purification and Characterization of Regulatory Subunit of Type I cAMP dependent Protein Kinase in E. coli |
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| LIU Xu Lei,LI Hui Ming,ZHAO Jian ,PEI Gang and MA Lan.Expression, Purification and Characterization of Regulatory Subunit of Type I cAMP dependent Protein Kinase in E. coli[J].Acta Biochimica et Biophysica Sinica,1999,31(4):459-462. |
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| Authors: | LIU Xu Lei LI Hui Ming ZHAO Jian PEI Gang and MA Lan |
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| Institution: | LIU Xu Lei,LI Hui Ming,ZHAO Jian 1,PEI Gang 1 and MA Lan * |
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| Abstract: | Regulatory subunit of type I cAMP dependent protein kinase (RI) possesses two cAMP binding sites with high affinity for cAMP. In the current study, human RI cDNA fragment coding for residue 93 381 was cloned from neuronal cells. After subcloning into pET30a, the recombinant RI was expressed in E.coli BL21. Considerable amount of soluble RI subunit was produced in BL21 upon induction of IPTG. RI was purified to homogeneity over 95% after His bind metal chelation chromatography. Approximately 8 mg pure RI was obtained from 0.1 liter of BL21 culture. Human RI prepared with this method binds to cAMP specifically and therefore can be used in cAMP assay. |
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| Keywords: | PKA cAMP expression |
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