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| 硫酸乙酰肝素3-O硫酸基转移酶5的表达、纯化及酶学性质研究 | |
| 引用本文： | 陕婧婧,窦文芳,李会,张旦旦,许泓瑜,陈敬华,许正宏.硫酸乙酰肝素3-O硫酸基转移酶5的表达、纯化及酶学性质研究[J].生物技术通报,2012(8):101-106. |
| 作者姓名： | 陕婧婧 窦文芳 李会 张旦旦 许泓瑜 陈敬华 许正宏 |
| 作者单位： | 1. 江南大学工业生物技术教育部重点实验室,无锡,214122 2. 江南大学医药学院制药工程研究室,无锡,214122 3. 江南大学工业生物技术教育部重点实验室,无锡214122;江南大学医药学院制药工程研究室,无锡214122 |
| 摘 要： | 经过PCR克隆得到硫酸乙酰肝素3-O硫酸基转移酶5(3-OST-5)的基因,将其与大肠杆菌表达载体pET-15b连接后,在大肠杆菌BL21(DE3)中诱导表达,使用镍亲和层析柱纯化得到具有活性的3-OST-5。经测定纯化后的3-OST-5比活达到0.58 U/mg,是纯化前的5.27倍,回收率达80.4%。在此基础上,研究了该酶的酶学性质,酶反应的最适温度为35℃,稳定范围为20-40℃;最适pH为7.0,在pH7.0-9.0范围内稳定。在反应液中加入终浓度为1 mmol/L的K+、Ca2+、Ba2+对酶促反应有一定的促进作用。 |
| 关 键 词： | 硫酸乙酰肝素 3-O硫酸基转移酶5 表达 纯化 酶学性质 |
Expression, Purification and Enzymatic Characterization of Heparan Sulfate 3-0 Sulfotransferase 5 |
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| Shan Jingjing , Dou Wenfang , Li Hui , Zhang Dandan , Xu Hongyu , Chen Jinghua , Xu Zhenghong.Expression, Purification and Enzymatic Characterization of Heparan Sulfate 3-0 Sulfotransferase 5[J].Biotechnology Bulletin,2012(8):101-106. | |
| Authors: | Shan Jingjing Dou Wenfang Li Hui Zhang Dandan Xu Hongyu Chen Jinghua Xu Zhenghong |
| Affiliation: | 1,2(1 The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122;2Laboratry of Pharmaceutical Engineering,School of Medicine and Pharmaceutics,Wuxi 214122) |
| Abstract: | The heparan sulfate 3-O sulfotransferase 5(3-OST-5) gene was amplified,and inserted into expression vector pET-15b,and then the plasmid pET15b/3-OST-5 was constructed and transformed into Escherichia coli BL21(DE3).SDS-PAGE showed that the gene 3-OST-5 was expressed successfully in recombinant E.coli BL21.Then 3-OST-5 was purified by Ni-NTA affinity chromatography.The results showed a single band about 31 kD on SDS-PAGE gel,and the specified activity was about 0.58 U/mg.The specified activity of 3-OST-5 is 5.27-fold higher than that of unpurified and the activity recovery is 80.4%.The optimum reaction temperature was 35℃,and the 3-OST-5 was more stable on the temperature of 20-40℃.The optimum reaction pH was 7.0,and the 3-OST-5 activity have little changed when incubated in the buffer of pH7.0-9.0.The addition of K+,Ca2+,Ba2+ with the final concentration of 1 mmol/L in the reaction solution plays a certain role in promoting the enzymatic reaction. |
| Keywords: | Heparan sulfate 3-O sulfotransferase 5 Expression Purification Enzymatic characterization |
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