高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (8): 1670-1674.doi: 10.7503/cjcu20140074

• 有机化学 • 上一篇    下一篇

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

侯欣彤1, 董媛1, 林瑞东1, 于梁1, 任媛媛1, 李剑光1, 高朝辉1,2,3(), 滕利荣1,2,3   

  1. 1. 吉林大学分子酶学工程教育部重点实验室, 2. 艾滋病疫苗国家工程实验室, 3. 生命科学学院, 长春 130012
  • 收稿日期:2014-01-23 出版日期:2014-08-10 发布日期:2014-03-31
  • 作者简介:高朝辉, 男, 博士, 副教授, 主要从事基因工程方面的研究. E-mail: chaohuigao@gmail.com
  • 基金资助:
    高等学校博士学科点专项科研基金新教师项目(批准号: 20100061120076)和中央高校基本科研业务费专项和林业公益性行业科研专项经费项目(批准号: 201204614)资助.

Gene Synthesis and Fusion Expression D-Aminoacylase Gene from Alcaligenes A-6

HOU Xintong1, DONG Yuan1, LIN Ruidong1, YU Liang1, REN Yuanyuan1, LI Jianguang1, GAO Chaohui1,2,3,*(), TENG Lirong1,2,3   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, 2. National Engineering Laboratory for AIDS Vaccine, 3. School of Life Science, Jilin University, Changchun 130012, China
  • Received:2014-01-23 Online:2014-08-10 Published:2014-03-31
  • Contact: GAO Chaohui E-mail:chaohuigao@gmail.com
  • Supported by:
    Supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20100061120076), the Fundamental Research Funds for the Central Universities and the Special Fund Project for the Scientific Research of the Forest Public Welfare Industry, China(No.201204614).

摘要:

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni2+柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.

关键词: D-氨基酰化酶, 产碱杆菌, 基因合成, 融合表达

Abstract:

The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E. coli., then the D-ANase gene was synthesized by the two-step method based on PCR technology. Synthetic gene and pET-32a vector were digested with BglⅡ and XhoⅠ, ligated by T4 DNA ligase. The ligation mixture transformed into E.coli BL21(DE3) competent cell. Recombinant protein was detected by SDS-PAGE, Western-blot and activity assay. D-ANase can be expressed efficiently in E. coli and the expressed protein content can reach to 69.2% of the total bacterial protein content. In addition, the fermentation activity can achieve 96 U/mL. After the ultrasonic cell disruption, the recombinant protein was purified by Ni2+ affinity chromatography column. The specific activity of the purified recombinant enzyme was 1692.3 U/mg and the purity could be up to 95%. Furthermore a firm foundation was laid for the industrial use of the D-ANase.

Key words: D-Aminoacylase, Alcaligenes A-6, Gene synthesis, Fusion expression

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