聚乙二醇修饰重组溶葡球菌酶的初步研究

中国生物工程杂志

2013, Vol. 33

Issue (6): 12-17

研究报告

聚乙二醇修饰重组溶葡球菌酶的初步研究

王永¹, 刘沐荣², 万海同¹, 刘翔², 张加慧², 封小燕², 张钒¹

1. 浙江中医药大学生物工程学院杭州 310053;
2. 杭州北斗生物技术有限公司杭州 310011

Chemical Modification of Lysostaphin with Activated Polyethlene Glycol

WANG Yong¹, LIU Mu-rong², WAN Hai-tong¹, LIU Xiang², ZHANG Jia-hui², FENG Xiao-yan², ZHANG Fan¹

1. Biological Engineering Institute of Zhejiang Chinese Medical University, Hangzhou 310053, China;
2. BIODOOR Biotechnology Co., Ltd of Hangzhou City in Zhejiang Province, Hangzhou 310011, China

全文: PDF(702 KB) HTML

摘要：

目的: 探讨聚乙二醇(PEG)修饰重组溶葡球菌酶(lysostaphin)的反应条件以及修饰后产物的纯化方法。方法: 采用超声波细胞粉碎机进行菌体破碎,阳离子交换层析、疏水层析进行蛋白纯化;在不同条件下,将活化的单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)与纯化后的lysostaphin反应,以单个PEG-Lysostaphin的比例为指标,用SDS-PAGE、MALDI-TOF-MS方法确定其在修饰产物中的所占比例;采用Sephacryl S-200分子筛凝胶层析法对修饰产物进行分离纯化。结果: mPEG-SPA修饰lysostaphin的反应条件为pH 8.0,温度4℃,lysostaphin与mPEG-SPA的质量比为1:5,反应时间2.0h;反应产物经一步Sephacryl S-200分子筛凝胶层析纯化后,初步实现分离。结论: 初步确定了聚乙二醇修饰lysostaphin的反应条件及修饰产物的纯化方法。

关键词： 聚乙二醇; 重组溶葡球菌酶; 纯化; 单甲氧基聚乙二醇琥珀酰亚胺丙酸酯

Abstract:

Objective: To study the reaction conditions for the chemical modification of recombinant lysostaphin (Lysostaphin) with activated polyethlene glycol (PEG) and the methods for the purification of PEG-Lysostaphin. Methods: Disrupt the cell wall of bacteria by ultrasonic cell disruptor and purify the lysostaphin by Cation exchange chromatography (SP) and Hydrophobic chromatography (HIC); Lysostaphin was chemical modified with activated mono-methoxy polyethlene glycol succinimide propionate (mPEG- SPA) under various conditions, the ration of mono-mPEG-Lysostaphin was determined by SDS-PAGE and EMALDI-TOF-MS; The reaction mixture was purified by Sephacryl S-200 molecular size exclusion chromatography. Results: The optimal reaction was pH 8.0,temperature 4℃, 1:5 for the mass ratio of lysostaphin and mPEG, two hours for reaction time was selected. The reaction mixture was preliminarily separated by one step Sephacryl S-200 molecular size exclusion chromatography. Conclusion: It’s preliminarily determined that the optimal reaction conditions for the chemical modification of lysostaphin with activated polyethlene glycol and the methods for the purification of PEG-Lysostaphin.

Key words: Polyethlene glycol (PEG) Recombinant lysostaphin Purification mPEG-SPA

收稿日期: 2013-01-29 出版日期: 2013-06-25

ZTFLH:

Q819

通讯作者: 王永 E-mail: wangyong19870602@126.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
	王永
	刘沐荣
	万海同
	刘翔
	张加慧
	封小燕
	张钒

引用本文:

王永, 刘沐荣, 万海同, 刘翔, 张加慧, 封小燕, 张钒. 聚乙二醇修饰重组溶葡球菌酶的初步研究[J]. 中国生物工程杂志, 2013, 33(6): 12-17.

WANG Yong, LIU Mu-rong, WAN Hai-tong, LIU Xiang, ZHANG Jia-hui, FENG Xiao-yan, ZHANG Fan. Chemical Modification of Lysostaphin with Activated Polyethlene Glycol. China Biotechnology, 2013, 33(6): 12-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I6/12

[1] Schindler C A, Schuhardt V T. Lysostaphin: A new bacteriolytic agent for the staphylococcus.Proc Natl Acad Sci U S A, 1964, 51(3): 414-421.
[2] Browder H P, Zygmunt W A, Young J R, et al. Lysostaphin: enzymatic mode of action. Biochem Biophys Res Commun, 1965, 19:383-389.
[3] Hrlary R Trayer, Buckley C E. Molecular properties of lysostaphin, a bacteriolytic agent specific for Staphylococcus aureus. the Journal of Biological Chemistry. 1970, 245(18), 4842-4846.
[4] Maria do Carmo de Freire Bastos, Bruna Goncalves Coutinho, Marcus Lívio Varella Coelho. Lysostaphin: A Staphylococcal bacteriolysin with potential clinical applications. Pharmaceuticals, 2010, 3:1139-1161.
[5] Michael W Climo, Kerstin Ehlert, Gordon L Archer. Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 2001, 45(5): 1431-1437.
[6] Paula Recsei, Alexandrad Gruss, Richard P Novick. Cloning,sequence,and expression of the lysostaphin gene from Staphylococcus simulans. Proc Natl Acad Sci USA, Biochemistry, 1987,84(5): 1127-1131.
[7] 杨信怡, 游雪甫, 蒋建东.溶葡球菌酶的研究进展.中国生化药物杂志,2005,26(6):372-374. Yang X Y,You X F,Jiang J D. Advances in lysostaphin research. Chinese Journal of Biochemical Pharmaceutics, 2005,26(6):372-374.
[8] Richard R Burgess, Murray P Deutscher. Guide to Protein Purification. 2^nd ed. Beijing: Science Press,2011,5:86-87.
[9] Walsh Scott,Anjali Shah, James Mond. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrobial Agents and Chemotherapy,2003,2:554-558.
[10] 吴宏, 房伟, 袁璟等. 溶葡球菌酶的定点突变与PEG巯基定点修饰.生物工程学报, 2011,27(11):1623-1630. Wu H, Fang W, Yuan J,et al. Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin. Chin J Biotech, 2011, 27(11): 1623-1630.
[11] Odintsov S G, Sabala I, Marcy Janiak M, et al. Latent LytM at 1. 3A resolution. J Mol Biol,2004, 16, 335(3): 775-785.

[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]	朱衡,张继福,张云,胡云峰. 环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶^*[J]. 中国生物工程杂志, 2020, 40(5): 57-68.
[7]	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.
[8]	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.
[9]	朱衡,张继福,张云,孙爱君,胡云峰. 聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 124-132.
[10]	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.
[11]	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.
[12]	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.
[13]	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.
[14]	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.
[15]	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.

Viewed

Full text

Abstract

Cited

Shared

Discussed