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Expression, purification and characterization of rat angiopoietin-2 in Pichia pastoris

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Abstract

Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.

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Acknowledgments

This work was financially supported by National Natural Science Foundation of China (No.30672403).

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Authors and Affiliations

  1. Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, 100850, Beijing, China

    Jinfeng Wang, Leilei Zhou, Xin Cai, Minji Zou, Yuanyuan Wang, Wenliang Fu, Jiaxi Wang & Donggang Xu

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  1. Jinfeng Wang
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  2. Leilei Zhou
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  3. Xin Cai
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  4. Minji Zou
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  5. Yuanyuan Wang
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  6. Wenliang Fu
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  7. Jiaxi Wang
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  8. Donggang Xu
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Corresponding author

Correspondence to Donggang Xu.

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J. Wang and L. Zhou contributed equally to this work as co-first authors.

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Wang, J., Zhou, L., Cai, X. et al. Expression, purification and characterization of rat angiopoietin-2 in Pichia pastoris . Mol Biol Rep 37, 3909–3913 (2010). https://doi.org/10.1007/s11033-010-0047-9

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  • DOI: https://doi.org/10.1007/s11033-010-0047-9

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