人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

中国生物工程杂志

2011, Vol. 31

Issue (12): 15-21

研究报告

人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

牛卫宁, 羊梦林, 曹珊珊, 许乐, 钦传光

西北工业大学生命学院西安 710072

Expression, Purification and Activity Assay of the Full-length and Truncated Human Cystathionine β-Synthase

NIU, Wei-ning, YANG Meng-lin, CAO Shan-shan, XU Le, QIN Chuan-guang

Faculty of Life Science, Northwestern Polytechnical University, Xi'an 710072, China

全文: PDF(549 KB) HTML

摘要：

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。

关键词： 人胱硫醚β-合酶; 大肠杆菌; 表达纯化; 酶活性测定; 胱硫醚β-裂解酶

Abstract:

The human cystathionine β-synthase(CBS) gene was ligated into vector pGEX-4T-1.The recombinant pGEX-4T-1-CBS was transformed into E.coli Rosetta (DE3), and the recombinant E.coli Rosetta (pGEX4T-1-CBS) strain which highly express CBS gene was constructed. After the recombinant E.coli was grown at 37℃ to an A₆₀₀ of 0.4~0.6, induced with IPTG at a final concentration of 0.1mmol/L for 16h at 30℃. The productivity of the soluble CBS reached 28mg/L. The supernatant of the disrupted cells by sonication was directly loaded on GSTrap Fast Flow, and the GST-CBS fusion protein was absorbed on the column. The GST-tagged CBS fusion protein bound to the column was treated with thrombin(10 unit of thrombin/mg protein) at 22℃ for 12-16h in the presence of 3% glycerol and 0.1% CHAPS. An easy one-step protocol to purify recombinant human CBS and in 54.8% overall yield had been used. From a 1L culture, some 15.2 mg of purified CBS protein at 95% purity as judged by SDS-PAGE could be abtained, and the purified enzyme had a specific activity of 143 unit/mg protein. S-adenosylmethionine(AdoMet) activates the CBS enzyme by as much as 5.1-fold in the presence of 1 mmol/L AdoMet with a specific activity of 735 unit/mg protein. Additionally, the recombinant E.coli Rosetta (pETDuet-1-CBS_1-413) strain which highly express truncated CBS(CBS_1-413) gene was constructed. Using a HisTrap Fast Flow affinity chromatography, the purity of recombinant CBS_1-413 lacking the C-terminal regulatory domain reached 95% by one-step purification with the specific activity of 965 unit/mg. The productivity of the soluble CBS reached 12.8mg/L and in 74.3% overall yield. In addition, The expression and purification of recombinant cystathionine β-lyase (CBL) in E.coli were described. The present study established a novel method, which relies on CBL as coupling enzyme, for detemination of CBS activity based on the color reaction between pyruvate and 2,4-dinitrophenylhydrazine.

Key words: Human cystathionine β-synthase E.coli Expression Activity assay Cystathionine β-lyase

收稿日期: 2011-05-20 出版日期: 2011-12-25

ZTFLH:

Q789

基金资助:

国家自然科学基金(20802057)、陕西省科技攻关计划(2011K08-12)资助项目

通讯作者: 牛卫宁:niuweining@nwpu.edu.cn E-mail: niuweining@nwpu.edu.cn

	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

引用本文:

牛卫宁, 羊梦林, 曹珊珊, 许乐, 钦传光. 人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定[J]. 中国生物工程杂志, 2011, 31(12): 15-21.

NIU, Wei-ning, YANG Meng-lin, CAO Shan-shan, XU Le, QIN Chuan-guang. Expression, Purification and Activity Assay of the Full-length and Truncated Human Cystathionine β-Synthase. China Biotechnology, 2011, 31(12): 15-21.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/15

[1] Yamanishi M, Kabil O, Sen S, et al. Structural insights into pathogenic mutations in heme-dependent cystathionine-β-synthase. J Inorg Biochem, 2006, 100: 1988-1995.

[2] Singh S, Madzelan P, Banejee R. Properties of an unusual heme cofactor in PLP-dependent cystathionine-β-synthase. Nat Prod Rep, 2007, 27: 631-639.

[3] Janosik M, Kery V, Gaustadnes M, et al. Regulation of human cystathionine β-synthase by s-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region. Biochemistry, 2001, 40:10625-10633.

[4] Oliveriusova J, Kery V, Maclean K N, et al. Deletion mutagenesis of human cystathionine β-synthase. J Biol Chem, 2002, 277(50): 48386-48394.

[5] Prudova A, Bauman Z, Braun A, et al. S-adenosylmethionine stabilizes cystathionine β-synthase and modulates redox capacity. PNAS, 2006,103:6489-6494.

[6] Taoka S, Ohja S, Shan X. et al., Evidence for heme-mediated redox regulation of human cystathionine beta-synthase activity. J Biol Chem,1998, 273:25179-25184.

[7] Frank N, Kent J O, Meier M, et al. Purification and characterization of the wild type and truncated human cystathionine β-Synthase enzymes expressed in E.coli. Arch Biochem Biophy, 2008,470: 64-72.

[8] Belew M S, Quazi F I, Willmore W G, et al. Kinetic characterization of recombinant human cystathionine-β-synthase purified from E.coli. Protein Expres Purif, 2009,64(2):139-145.

[9] 郑斌,韩梅,温进坤. 胱硫醚β-合酶活性的测定及应用. 中国动脉硬化杂志, 1999,7(4):351-353 Zheng B, Han M, Wen J K. Chin J Arterioscler, 1999,7(4):351-353.

[10] 李丽帆,方显明,顾国龙,等. 胱硫醚β-合酶活性测定及应用. 第四军医大学学报, 2009, 30(9):863-864 Li L F, Fang X G, Gu G L, et al. J Fourth Mil Med Univ, 2009, 30(9):863-864.

[11] 胡晓冰,林标声,杨生玉,等. 测定丙酮酸发酵生产产量的几种化学方法的比较. 粮油加工,2010, 12:165-168. Hu X B, Lin B S, Yang S Y, et al. Cereals and Oils Processing, 2010, 12:165-168.

[12] Kery V, Elleder D, Kraus J P. δ-Aminolevulinate increases heme saturation and yield of human cystathionine β-Synthase expressed in Escherichia coli. Arch Biochem Biophys, 1995, 316(1):24-29.

[1]	乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析^*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[2]	何若昱,林福玉,高向东,刘金毅. 信号肽在大肠杆菌分泌系统中的研究与应用进展[J]. 中国生物工程杂志, 2021, 41(5): 87-93.
[3]	吴弘轩, 杨金花, 沈培杰, 李清晨, 黄建忠, 祁峰. 利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究 ^*[J]. 中国生物工程杂志, 2021, 41(1): 12-19.
[4]	闫伟欢,黄统,洪解放,马媛媛. 丁醇在大肠杆菌中的生物合成研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 69-76.
[5]	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.
[6]	童梅,程永庆,刘金毅,徐晨. 促进大肠杆菌周质空间小分子抗体表达的菌种构建方法^*[J]. 中国生物工程杂志, 2020, 40(5): 48-56.
[7]	杨丽,石晓宇,李文蕾,李剑,徐寒梅. 构建噬菌体展示抗体库过程中电穿孔法的条件优化[J]. 中国生物工程杂志, 2020, 40(4): 42-48.
[8]	乐易林,傅毓,倪黎,孙建中. 热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 72-78.
[9]	杭海英,刘春春,任丹丹. 流式细胞术的发展、应用及前景 ^*[J]. 中国生物工程杂志, 2019, 39(9): 68-83.
[10]	赵程程,孙长坡,常晓娇,伍松陵,林振泉. 大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(4): 69-77.
[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]	贺雪婷,张敏华,洪解放,马媛媛. 大肠杆菌丁醇耐受机制及耐受菌选育研究进展 ^*[J]. 中国生物工程杂志, 2018, 38(9): 81-87.
[14]	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.
[15]	胡立强, 郑文, 钟艺, 杜丹, 杨浩, 龚萌. 抗病毒蛋白RC28在大肠杆菌和毕赤酵母中的表达及活性比较[J]. 中国生物工程杂志, 2017, 37(1): 14-20.

Viewed

Full text

Abstract

Cited

Shared

Discussed