Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli

SU Nan; WU Jingjun; LI Ye; ZHANG Chong; LI Mei; XING Xinhui

doi:10.3969/j.issn.0438-1157.2014.07.045

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Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli

更新时间：2025-12-10

- Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli
- CIESC Journal Issue 7, Pages: 2829-2842(2014)
- 作者机构：
  
  1. 清华大学化学工程系, 工业生物催化教育部重点实验室, 生物化工研究所,北京,100084
  2. 北京电子科技职业技术学院生物技术系,北京,100029
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.0438-1157.2014.07.045
  CLC： Q028.8
- Online First：05 July 2014，
  
  Published：05 July 2014
- 稿件说明：
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SU Nan, WU Jingjun, LI Ye, et al. Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli[J]. CIESC Journal, 2014, (7): 2829-2842.
DOI：

SU Nan, WU Jingjun, LI Ye, et al. Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli[J]. CIESC Journal, 2014, (7): 2829-2842. DOI： 10.3969/j.issn.0438-1157.2014.07.045.

摘要

肝素酶Ⅲ（heparinase Ⅲ，HepC）是一种重要的多糖裂解酶，在抗癌药物开发、低分子量肝素生产以及肝素类药物的质量控制等方面具有重要的作用。目前制约其工业应用前景的主要技术瓶颈是生产成本高，异源重组表达效果差，缺乏高效的表达方法。本研究借鉴前期经验，通过HepC基因的密码子优化，并与麦芽糖结合蛋白（maltose-binding protein，MBP）融合，构建了MBP-coHepC（基因密码子优化后的蛋白为coHepC）的大肠杆菌表达体系，结合培养条件优化大幅度提高了HepC的可溶表达比例，其摇瓶发酵总酶活值达到7603.46 IU·L-1；同时，本研究从转录和翻译水平揭示了HepC表达效果提高的可能原因。这些研究为肝素酶Ⅲ的应用发展提供了基础。

Abstract

Heparinase Ⅲ (HepC) is an important polysaceharide lyase

which plays a vital role in the development of anticancer drugs

production of low molecular weight heparin(LMWH)and quality control of the heparins. The main bottlenecks of HepC application are high production cost

poor level of heterologous recombinant expression as well as lack of efficient expression system. Based on the previous experience

the codon optimization of HepC gene (the optimized gene product named as coHepC) and E.coli expression system by constructing the MBP-coHepC fusion protein were studied. Combining with further optimization of the culture conditions

the soluble expression ratio of the fusion protein was significantly increased and the total enzyme activity in the shake-flask culture reached 7603.46 IU·L-1. At the same time

the possible reasons for the HepC improved expression were analyzed at transcription and translation levels of HepC gene. These studies provided the basis for applications of heparinase Ⅲ.

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