| 大鼠Gs alpha亚基的原核表达和纯化 |
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| 引用本文： | 施巍炜,黄有国,孙伟. 大鼠Gs alpha亚基的原核表达和纯化[J]. 中国生物化学与分子生物学报, 2000, 16(1): 28-31 |
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| 作者姓名： | 施巍炜 黄有国 孙伟 |
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| 作者单位： | 1. 生物大分子国家重点实验室,中国科学院生物物理研究所,北京,100101
2. 青岛大学医学院附属医院,青岛,266021 |
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| 基金项目： | 中科院重大项目!( KJ95 1-B1-6 0 9)，国家自然科学基金委重点项目!( 3 973 0 13 0 )资助 |
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| 摘 要： | 用 PCR的方法 ,在大鼠 Gs alpha亚基的 C端引入了 6个外源组氨酸 (即 6×His- Tag)并以此为纯化标记 .构成的表达载体 p QE60 /rat Gsα( L)在大肠杆菌 BL2 1 ( DE3)中获得了稳定的表达 .经 DEAE- Sephacel离子交换柱和 Ni- NTA Agarose亲和层析获得纯化的具有较高 [35S]- GTPγS结合活力的重组大鼠 Gs alpha亚基
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| 关 键 词： | 大鼠Gsalpha亚基 6×His-Tag 表达 [~(35)S]-GTP_γS结合活力 |
| 收稿时间： | 2000-02-20 |
Prokaryotic Expression and Purification of Rat Gs alpha Subunit |
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| SHI Wei-wei,HUANG You-guo,SUN Wei. Prokaryotic Expression and Purification of Rat Gs alpha Subunit[J]. Chinese Journal of Biochemistry and Molecular Biology, 2000, 16(1): 28-31 |
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| Authors: | SHI Wei-wei HUANG You-guo SUN Wei |
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| Affiliation: | (National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101) SUN Wei (Qingdao Medical College,Qingdao 266021 |
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| Abstract: | In an attempt to facilitate purification,six histidines were added by PCR as purification tag to the C terminal of rat Gs alpha subunit.Both the constructed expression plasmid (pQE60/rat Gsα(L)) and helper plamid (pREP4) were transformed into E.coli BL21(DE3).At low temperature (30℃) and low IPTG (30 μmol/L), the recombinant rat Gsα was stably expressed.The target protein was purified by DEAE Sephacel ion exchange column and Ni NTA Agarose affinity column.The purified recombinant protein showed an apparent molecular weight of 52 kD examined by SDS PAGE with silver staining and had higher [ 35 S] GTP γS binding activity. |
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| Keywords: | Rat Gs alpha subunit 6×His Tag Expression [ 35 S] GTP γS binding activity |
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