| P450cam突变体蛋白表达、纯化与结晶的改进及其四突变体（F87L/Y96F/L244A/V247A）晶体结构 |
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| 引用本文： | 刘斌斌,孙蕾,徐峰,娄智勇,庞海,L.L.WONG,刘一苇.P450cam突变体蛋白表达、纯化与结晶的改进及其四突变体（F87L/Y96F/L244A/V247A）晶体结构[J].生物化学与生物物理进展,2004,31(7):611-615. |
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| 作者姓名： | 刘斌斌 孙蕾 徐峰 娄智勇 庞海 L.L.WONG 刘一苇 |
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| 作者单位： | 1. 清华大学结构生物学实验室,教育部蛋白质科学重点实验室,北京,100084
2. Department of Chemistry Inorganic Chemistry Laboratory University of Oxford U.k |
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| 摘 要： | 在P450cam突变体蛋白纯化过程中使用280 nm/392 nm双波长比值检测蛋白质纯度, 采用β-巯基乙醇对蛋白质进行还原性保护，有效地缩短了纯化进程，纯化效率相应提高.以PEG 8000为沉淀剂经悬滴汽相扩散法筛选得到适合衍射的P450cam四突变体（F87L/Y96F/L244A/V247A）晶体，在Mar-Research面探测器系统上收集了0.22 nm分辨率的X射线衍射数据.采用同晶差值傅立叶法解析结构，最后的晶体学R因子和Rfree分别为0.197和0.247，键长偏差为0.001 77 nm，键角偏差为1.96°.结构测定显示P450cam四突变体（F87L/Y96F/L244A/V247A）和P450cam野生型的整体构象无重大变化，突变后活性口袋变大而疏水性增加，这与突变设计预期目标一致.
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| 关 键 词： | P450cam，四突变体，纯化，双波长检测，晶体结构 |
| 收稿时间： | 1/7/2004 12:00:00 AM |
| 修稿时间： | 2004/2/28 0:00:00 |
The Improved Procedure for The Expression, Purification and Crystallization of Mutants of Cytochrome P450cam and The Crystal Structure of The F87L/Y96F/L244A/V247A Mutant |
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| LIU Bin-Bin,SUN Lei,XU Feng,LOU Zhi-Yong,PANG Hai,L.L.WONG and LIU Yi-Wei.The Improved Procedure for The Expression, Purification and Crystallization of Mutants of Cytochrome P450cam and The Crystal Structure of The F87L/Y96F/L244A/V247A Mutant[J].Progress In Biochemistry and Biophysics,2004,31(7):611-615. |
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| Authors: | LIU Bin-Bin SUN Lei XU Feng LOU Zhi-Yong PANG Hai LLWONG and LIU Yi-Wei |
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| Institution: | Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China;Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China;Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China;Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China;Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China;Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, U.K.;Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology, Department of Biological Science and Biotechnology ,Tsinghua University, Beijing 100084, China |
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| Abstract: | |
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| Keywords: | P450cam four mutants purification double UV check crystal structure |
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