人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

doi:10.13523/j.cb.20150805

中国生物工程杂志

2015, Vol. 35

Issue (8): 30-37 DOI: 10.13523/j.cb.20150805

研究报告

人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

黄鹏¹, 李文姝³, 谢君⁴, 鲍建瑛¹, 曹晓娥¹, 余龙⁴, 徐一新²

1. 上海健康医学院基础医学院上海 200237;
2. 上海健康医学院药学院上海 200237;
3. 上海纽约大学文理学部上海 200122;
4 复旦大学遗传工程国家重点实验室上海 200433

Expression of Human Lysozyme-like Protein 6 in Pichia pastoris and Analysis of Enzymatic Activity of the Protein

HUANG Peng¹, LI Wen-shu³, XIE Jun⁴, BAO Jian-ying¹, CAO Xiao-e¹, YU Long⁴, XU Yi-xin²

1. College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 200237, China;
2. College of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai 200237, China;
3. College of Arts and Sciences, New York University, Shanghai 200122, China;
4. State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, China

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摘要：

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein 6,hLyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成hLyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的pPIC9K质粒构建重组表达载体pPIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDS-PAGE检测在14.8 kDa处有重组hLyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定hLyzl6酶学活性,结果表明hLyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适pH为5.5,其酶活力为54 700U/mg,Cu²⁺对其活性有明显抑制,EC₅₀为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组hLyzl6,证实其在体外具有杀菌活性,初步揭示hLyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究hLyzl6的功能和应用开发奠定了基础。

关键词： 人源类溶菌酶蛋白6; 毕赤酵母; 表达; 杀菌活性

Abstract:

The aim is to realize the recombinant expression of human lysozyme-like protein 6 (hLyzl6) by the Pichia pastoris expression system and to investigate its enzymatic activity. The hLyzl6 gene was synthesized chemically according to the codon usage preference of Pichia pastoris and cloned into the expression vector pPIC9K. The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115. After screening by G418 plate, the yeast strain was cultured and induced with methanol. The recombinant protein was purified by chitin affinity chromatography and subjected to activity assay. The results showed that the lytic activity in the culture supernatant reached the peak after 72 h of induction and the existence of the recombinant protein was confirmed by SDS-PAGE. The recombinant hLyzl6 exhibited bactericidal activity against Micrococcus lysodeikticus by using turbidimetric assay and had the activity of 54 700 U/mg. The optimum temperature and pH for hLyzl6 were 40℃ and 5.5 respectively and Cu²⁺ was found to have strong inhibition effect on hLyzl6. The EC₅₀ value was 30.2799 mg/L. In conclusion, recombinant hLyzl6 was expressed successfully by the Pichia pastoris strain GS115 and had in vitro bactericidal activity, which indicated that hLyzl6 may play a role in innate immunity of the male reproductive system and laid a foundation for its functional research and application.

Key words: Human lysozyme-like protein 6 Pichia. pastoris Expression Bactericidal activity

收稿日期: 2015-07-10 出版日期: 2015-08-25

ZTFLH:

Q819

基金资助:

上海市自然科学基金(15ZR1421800)、上海市高校青年教师资助培养计划(PF-JZ-QT02022016)资助项目

通讯作者: 余龙,徐一新 E-mail: longyu@fudan.edu.cn;xuyixin1968@163.com

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引用本文:

黄鹏, 李文姝, 谢君, 鲍建瑛, 曹晓娥, 余龙, 徐一新. 人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析[J]. 中国生物工程杂志, 2015, 35(8): 30-37.

HUANG Peng, LI Wen-shu, XIE Jun, BAO Jian-ying, CAO Xiao-e, YU Long, XU Yi-xin . Expression of Human Lysozyme-like Protein 6 in Pichia pastoris and Analysis of Enzymatic Activity of the Protein. China Biotechnology, 2015, 35(8): 30-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150805 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/30

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