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Construction of recombinant plasmid and prokaryotic expression in E. coli and biological activity analysis of human
Jin-Ping Zheng1, Hai-Ying Tang, Xian-Jiu Chen
1Department of Toxicology, Shanxi Medical Universiy, Taiyuan 030001, China. zhengzheng200101@yahoo.com.cn
Background:
The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten.
Methods:
The arresten gene was obtained from a healthy puerperal's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5alpha, BL21 and BL21 (DE3) by the CaCl2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purified, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM).
Results:
The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities.
Conclusions:
The successful construction of recombinant plasmid pBV220/arr and the effective expression in E.coli have laid a foundation for further study of its anti-angiogenic function and may pave the way for future anti-tumor application.
Insights
Arresten, an angiogenesis inhibitor, was successfully cloned and expressed in E. coli. This research lays the groundwork for developing new anti-cancer therapies by targeting tumor blood vessel formation.
Area of Science:
- Molecular Biology
- Biotechnology
- Cancer Research
Background:
- Cancer proliferation and metastasis are angiogenesis-dependent.
- Angiogenesis inhibitors offer a viable cancer treatment strategy.
- Arresten shows potential as a novel angiogenesis inhibitor.
Purpose of the Study:
- To assess the anti-angiogenic activity of arresten.
- To clone and express the arresten gene in a prokaryotic system.
Main Methods:
- Arresten gene isolated via RT-PCR from placenta.
- Molecular cloning into pBV220 plasmid and transformation into E. coli strains.
- Expression analysis using SDS-PAGE, purification, and biological activity testing on chorioallantoic membrane (CAM).
Main Results:
- Recombinant plasmid pBV220/arr successfully constructed.
- High-level expression of arresten protein achieved in E. coli BL21 (DE3).
- Expressed arresten protein (26 kDa) demonstrated significant anti-angiogenic activity.
Conclusions:
- Successful construction and expression of arresten provide a basis for further research.
- The findings support arresten's potential as an anti-angiogenic agent.
- This work may lead to future anti-tumor therapeutic applications.
