| 人源Fank1蛋白质FN3结构域多肽的原核表达，纯化及晶体筛选 |
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| 引用本文： | 黄海涛,宋伟,缪时英,王琳芳.人源Fank1蛋白质FN3结构域多肽的原核表达，纯化及晶体筛选[J].国外医学：分子生物学分册,2009(3):193-196. |
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| 作者姓名： | 黄海涛 宋伟 缪时英 王琳芳 |
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| 作者单位： | 中国医学科学院基础医学研究所医学分子生物学国家重点实验室,北京市100005 |
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| 基金项目： | 国家重点基础研究发展项目（973）计划（No.2006CB504001）,重大研究计划项目（No.2006CB944002） |
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| 摘 要： | 目的纯化人源Fank1（fibronectin type Ⅲ and ankyrin repeat domain1）蛋白质N端FN3（fibronectin typeⅢ，Ⅲ型纤黏连蛋白）结构域蛋白，用于晶体生长的三维结构分析。方法将FN3结构域基因片段克隆至原核表达载体pGEX-6P-1中，将菌落PCR和测序鉴定正确的重组质粒转化E.coli BL21（DE3）后获得表达菌株。该菌株经IPTG诱导高效表达出带有GST标签的可溶性的融合蛋白，经过Glutathione Sepha-rose^TM 4B亲和层析、Hiload16／60 superdex200分子筛层析纯化后，蛋白纯度达到95％以上。结果纯化蛋白采用悬滴气相扩散法得到棒状晶体。结论成功制备了高纯度FN3蛋白，获得FN3蛋白质晶体，为进一步的三维结构解析及Fank1功能研究奠定了基础。
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| 关 键 词： | Fankl FN3结构域 表达 蛋白质纯化 晶体生长 |
Prokaryotic Expression,Purification and Crystallization of Human Fank1 Protein FN3 Domain |
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| Authors: | HUANG Haitao SONG Wei MIAO Shiying WANG Linfang |
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| Institution: | (National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing, 100005, China) |
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| Abstract: | Objective To obtain human Fank1 protein FN3 domain for X-ray diffraction exper-iments. Methods The gene of FN3 domain from FANK1 was subcloned into expression vector pGEX-6P-1 and overexpressed in E. coli BL21（DE3） . FN3 protein was purified with Glutathione Sepharose^TM4B affinity followed by gel filtration chromatography. Results A large amount of FN3 protein with stick-like crystals was obtained. Conclusion Application of the obtained crystals for X-ray diffraction experiments may contribute to structural research of FN3 and functional illustration of Fank1 protein. |
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| Keywords: | Fank1 FN3 domain prokaryotic expression purification crystallization |
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