Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.
Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.
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来自超嗜热菌 Aquifex aeolicus 和 Archaeoglobus fulgidus 的烷基化损伤修复蛋白 O6-烷基鸟嘌呤-DNA 烷基转移酶。
DOI：
10.1042/bj20030809
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Pegg,AnthonyE
中科院分区：
文献类型：
--
作者：
Kanugula,Sreenivas;Pegg,AnthonyE
AGT（O 6-alkylguanine DNA alkyltransferase）是一种重要的DNA修复蛋白，可保护细胞免受烷化剂的杀伤和诱变。从两个极端嗜热生物，细菌Aquifex aeolicus和古细菌Archaeoglobus fulgidus的AGT基因PCR衍生和克隆到表达载体。Aq. aeolicusAGT基因编码一个201个氨基酸的蛋白质，分子量为23000 Da和Ar。fulgidusAGT编码147个氨基酸的蛋白质，分子量为16718 Da。aeolicusandAr. fulgidusAGTs在大肠杆菌中高水平表达，融合到N-末端多组氨酸标签上，允许通过金属亲和层析进行单步分离和纯化。两种AGT均形成包涵体，并且在天然纯化条件下不可溶。因此，在8.0 M尿素存在下，在蛋白质变性条件下进行AGT分离。在小牛胸腺DNA存在下，通过复性AGT获得可溶性AGT。这两种AGTs在修复DNA中的O 6-甲基鸟嘌呤和O 4-甲基胸腺嘧啶方面都有活性，但修复率较低。它们表现出热稳定性和最佳活性在高温下。热稳定AGTs，特别是来自Aq. aeolicus，很容易被低分子量的α-O 6-苄基鸟嘌呤灭活，这是目前在临床试验中，以加强癌症化疗。
AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. TheAGTgenes from two extremely thermophilic organisms, the bacteriumAquifex aeolicusand the archaeonArchaeoglobus fulgiduswere PCR-derived and cloned into an expression vector. The nucleotide sequence of theAq. aeolicusAGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da andAr. fulgidusAGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. TheAq. aeolicusandAr. fulgidusAGTs were expressed at high levels inEscherichia colifused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairingO6-methylguanine and, at a lower rate,O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that fromAq. aeolicus, were readily inactivated by the low-molecular-mass inhibitorO6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.
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DOI：
10.1021/bi982908w
发表时间：
1999
期刊：
Biochemistry.
影响因子：
--
作者：
Spratt,TE;Wu,JD;Levy,DE;Kanugula,S;Pegg,AE
通讯作者：
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3.9
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影响因子：
11.5
作者：
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影响因子：
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作者：
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