Thermal Shift Assays在重组森林脑炎病毒丝氨酸蛋白酶纯化中的应用

中国生物工程杂志

2009, Vol. 29

Issue (11): 53-59

技术与方法

Thermal Shift Assays在重组森林脑炎病毒丝氨酸蛋白酶纯化中的应用

朱俊萍**,范东瀛

首都医科大学基础医学院北京 100069

Application of Thermal Shift Assays in Purification of Recombinant TBEV Serine Protease

ZHU Jun-ping,FAN Dong-ying

School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

全文: PDF(1367 KB) HTML

摘要：

应用Thermal Shift Assays技术，高通量优化了重组森林脑炎病毒丝氨酸蛋白酶纯化过程中所使用的缓冲液中盐的种类和pH，显著改善了该重组蛋白在纯化过程中出现可溶性聚集体的现象。最终获得的重组蛋白质单体量由原先的75%提高到99%以上，极大提高了该纯化蛋白质的均一性和质量，为后续的蛋白质结晶研究奠定了基础。

关键词： Thermal Shift Assays; 重组蜱传播脑炎病毒丝氨酸蛋白酶; 纯化

Abstract:

Thermal Shift Assays was applied for high-throughput screening optimized buffer system used in the gel filtration chromatography for purification of recombinant TBEV serine protease. The optimization included kinds of salt and pH. It was optimized new buffer remarkably improved the quality of the purified TBEV serine protease by increasing its monomer from 75% to 99%. This result may improve future crystal studies of this protein.

Key words: Thermal Shift Assays Recombinant TBEV serine protease Purification

收稿日期: 2009-07-08 出版日期: 2009-12-07

ZTFLH:

Q789

基金资助:

京市属高等学校人才强教计划资助

通讯作者: 朱俊萍 E-mail: jpzhu0217@vip.sina.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	朱俊萍
	范东瀛

引用本文:

朱俊萍范东瀛. Thermal Shift Assays在重组森林脑炎病毒丝氨酸蛋白酶纯化中的应用[J]. 中国生物工程杂志, 2009, 29(11): 53-59.

SHU Dun-Ping, FAN Dong-Ying. Application of Thermal Shift Assays in Purification of Recombinant TBEV Serine Protease. China Biotechnology, 2009, 29(11): 53-59.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I11/53

［1］	Mezzasalma T M, Kranz J K, Chan W, et al. Enhancing recombinant protein quality and yield by protein stability profiling. J Biomol Screen, 2007,12(3):418～428
［2］	Chi E Y, Krishnan S, Randolph T W, et al. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res,2003,20(9):1325～1336
［3］	Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res,2008,25(7):1487～1499
［4］	Geerlof A, Brown J, Coutard B, et al. The impact of protein characterization in structural proteomics. Acta Cryst,2006,D62: 1125～1136
［5］	Nettleship J E, Brown J, Groves M R, et al. Structural Proteomics. Humana Press,2008,426:299～318
［6］	El Omari K, Bird L E, Nichols C E, et al. Crystal structure of CC3(TIP30): implications for its role as a tumor suppressor. J Biol Chem,2005,280: 18229～18236
［7］	Ericsson U B, Hallberg B M, Detitta G T, et al. Thermofluorbased highthroughput stability optimization of proteins for structural studies. Anal Biochem,2006,357(2):289～298
［8］	Majava V, L?ytynoja N, Chen W Q, et al. Crystal and solution structure, stability and posttranslational modifications of collapsin response mediator protein 2. FEBS J,2008,275(18):4583～4596
［9］	Maryanoff B E, McComsey D F, Lee J, et al. Carbonic anhydraseII inhibition. what are the true enzymeinhibitory properties of the sulfamide cognate of topiramate. J Med Chem,2008,51(8):2518～2521
［10］	Cummings M D, Farnum M A, Nelen M I, et al. Universal screening methods and applications of ThermoFluor. J Biomol Screen,2006,11(7):854～863
［11］	Carver T E, Bordeau B, Cummings M D, et al. Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology. J Biol Chem,2005,280(12):11704～11712
［12］	Kean J, Cleverley R M, O'Ryan L, et al. Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluorbased stability assay. Mol Membr Biol,2008,25(8):653～663

[1]	张玲,曹小丹,杨海旭,李文蕾. 连续流层析技术在亲和层析中的应用及生产放大评估[J]. 中国生物工程杂志, 2021, 41(6): 38-44.
[2]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[3]	蒋丹丹,王云龙,李玉林,张怡青. 含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 22-29.
[4]	谢航航,白红妹,叶超,陈永俊,袁明翠,马雁冰. 易发生聚集的重组HBcAg病毒样颗粒的纯化^*[J]. 中国生物工程杂志, 2020, 40(5): 40-47.
[5]	位薇,常保根,王英,路福平,刘夫锋. Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证^*[J]. 中国生物工程杂志, 2020, 40(5): 22-29.
[6]	刘珍珍,田大勇. 狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 ^*[J]. 中国生物工程杂志, 2020, 40(4): 25-33.
[7]	朱彤彤,杨磊,刘应保,孙文秀,张修国. 辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 116-123.
[8]	潘炳菊,张宛怡,申会涛,刘婷婷,李中媛,罗学刚,宋亚囝. 甘露寡糖分离纯化研究进展^*[J]. 中国生物工程杂志, 2020, 40(11): 90-95.
[9]	谢玉锋,韩雪梅,路福平. 副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 ^*[J]. 中国生物工程杂志, 2019, 39(5): 72-79.
[10]	付大伟,孙莹莹,徐伟. 融合蛋白NusA-hRI的高效异源表达、纯化及活性分析[J]. 中国生物工程杂志, 2019, 39(3): 21-28.
[11]	景佳美,徐欣,王敏,彭如超,施一. 沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 ^*[J]. 中国生物工程杂志, 2019, 39(12): 18-23.
[12]	朱梦露,王雪雨,刘鑫,路福平,孙登岳,秦慧民. 一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2019, 39(12): 24-34.
[13]	童超迪,吴坚平,杨立荣,徐刚. X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究 ^*[J]. 中国生物工程杂志, 2018, 38(8): 19-25.
[14]	陈军军,娄颖,张元兴,刘琴,刘晓红. 增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化 ^*[J]. 中国生物工程杂志, 2018, 38(7): 14-20.
[15]	李诗洁,杨艳坤,刘萌,白仲虎,金坚. SUMO蛋白酶Ulp1的高效表达纯化并通过His-SUMO标签制备scFv ^*[J]. 中国生物工程杂志, 2018, 38(3): 51-61.

Viewed

Full text

Abstract

Cited

Shared

Discussed