栖热菌噬菌体TSP4 dCTP脱氨酶基因的克隆表达

中国生物工程杂志

2013, Vol. 33

Issue (4): 34-39

研究报告

栖热菌噬菌体TSP4 dCTP脱氨酶基因的克隆表达

高婷婷, 张琦, 魏云林, 季秀玲, 林连兵

昆明理工大学生命科学与工程学院昆明 650500

Cloning and Expression of the dCTP Deaminase Gene from Thermus Phage TSP4

GAO Ting-ting, ZHANG Qi, WEI Yun-lin, JI Xiu-ling, LIN Lian-bing

College of Life Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China

全文: PDF(545 KB) HTML

摘要： 将编码栖热菌噬菌体TSP4菌株的dCTP脱氨酶tspdCD基因亚克隆到表达载体pET-32a中,并将重组质粒pET-32a -tspdCD转化至大肠杆菌Rosetta(DE3)中进行诱导表达。SDS-PAGE分析结果显示,目的蛋白经IPTG诱导后在大肠杆菌Rosetta(DE3)中以可溶性形式高效表达。通过Ni-NTA agarose亲和层析柱纯化表达的tspdCD,并对其活性,最适作用温度、pH、底物特异性以及金属离子和有机溶剂对其活性的影响进行测定,检测结果表明重组蛋白活性达到4.12U/mg,它的最适作用温度和pH分别为60℃和7.5,最佳反应底物是dCTP,2mmol/L的Ca²⁺和Mg²⁺对其活性具有明显的促进作用,而同浓度的 Ni²⁺ 和Cu²⁺对其活性产生了明显的抑制作用,10%(V/V)的乙酸乙酯和异丙醇对其活性有很明显的促进作用,而同体积比的丙酮对其活性产生明显的抑制作用。实现了tspdCD在大肠杆菌系统中的功能性表达,为进一步研究高温噬菌体tspdCD的功能奠定了基础。

关键词： 栖热菌噬菌体; tspdCD; 克隆表达; 亲和纯化

Abstract: The gene encoding a dCTP deaminase tspdCD from Thermus phage TSP4 was subcloned into pET-32a, generating a recombinant plasmid pET-32a-tspdCD which was further transformed into Escherichia coli Rosetta(DE3) for expression. Analysis of SDS-PAGE showed that target protein was highly expressed as a soluble form after IPTG induction. The expressed protein was purified using Ni-NTA agarose, which was further used to analyze the enzyme activities including optimum activity temperature, pH and substrate, as well as the effects of metal ions and organic solvents on the enzyme activity. The results showed that the specific activity of the expressed tspdCD was 4.12U/mg. Its optimum activity temperature and pH were 60℃ and 7.5, repectively, and its optimum substrate was dCTP. The enzyme activity was stimulated by Ca²⁺ and Mg²⁺ of 2mmol/L, but was inhibited by Ni²⁺ and Cu²⁺ of 2 mmol/L. The results also showed that 10%(V/V) ethyl acetate and isopropanol significantly increased the activity of the expressed tspdCD while 10%(V/V) acetone had a significant inhibitory effect for the same enzyme. The successful expression of the phage tspdCD, in E. coli provides a good basis for further functional study of the purified enzyme.

Key words: Thermus phage tspdCD Cloning and expression Affinity purification

收稿日期: 2012-12-17 出版日期: 2013-04-25

ZTFLH:

Q786

基金资助: 国家自然科学基金资助项目(30960022,31160035)

通讯作者: 林连兵 E-mail: linlb@sohu.com

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引用本文:

高婷婷, 张琦, 魏云林, 季秀玲, 林连兵. 栖热菌噬菌体TSP4 dCTP脱氨酶基因的克隆表达[J]. 中国生物工程杂志, 2013, 33(4): 34-39.

GAO Ting-ting, ZHANG Qi, WEI Yun-lin, JI Xiu-ling, LIN Lian-bing. Cloning and Expression of the dCTP Deaminase Gene from Thermus Phage TSP4. China Biotechnology, 2013, 33(4): 34-39.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/34

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