Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF.
Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF.
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钴胺素（维生素 B12）生物合成——巨大芽孢杆菌 S-腺苷-L-甲硫氨酸依赖性钴-前皮质素-4 转甲基酶 CbiF 的克隆、表达和结晶。
DOI：
10.1046/j.1432-1327.1998.2540341.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Warren,MJ
中科院分区：
文献类型：
--
作者：
Raux,E;Schubert,HL;Woodcock,SC;Wilson,KS;Warren,MJ
巨大芽孢杆菌 cbiF 编码厌氧钴胺素生物合成途径的钴-前皮质素-4S-腺苷-L-甲硫氨酸依赖性转甲基酶，已在大肠杆菌中克隆并过表达为带有组氨酸标签的重组蛋白。通过金属螯合色谱和高分辨率阴离子交换色谱的组合将蛋白质纯化至均质。通过 SDS/PAGE 检测，该蛋白迁移的亚基质量为 31 kDa；通过分析凝胶过滤，该蛋白的分子量为 62 kDa，表明天然重组蛋白是同源二聚体。 His 标记蛋白具有生理活性，因为它能够补充鼠伤寒沙门氏菌 cbiF 突变体。然而，该蛋白质与 S-腺苷-L-甲硫氨酸的结合力与其他 Corrin 生物合成转甲基酶的结合力不同。纯化蛋白质的结晶筛选鉴定出两种离散的晶体形式。其中一种形式已被表征并收集了完整的数据集。
TheBacillus megaterium cbiF, encoding the cobalt‐precorrin‐4S‐adenosyl‐L‐methionine‐dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His‐tagged recombinant protein inEscherichia coli. The protein was purified to homogeneity by a combination of metal chelate chromatography and high‐resolution anion‐exchange chromatography. The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer. The His‐tagged protein was physiologically active as it was able to complement aSalmonella typhimurium cbiFmutant. However, the protein did not bindS‐adenosyl‐L‐methionine with the same avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identification of two discrete crystal forms. One of these forms has been characterised and a full data set collected.