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Prokaryotic expression, purification and functional characterization of recombinant human RIP2

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An Erratum to this article was published on 28 November 2012

Abstract

Receptor-interacting protein 2 (RIP2) is a member of the receptor interacting protein (RIP) family and plays an important role in the innate and adaptive immune responses. Overexpression of RIP2 mediates divergent signaling pathways including NF-κB activation and cell death. To further investigate the biological activity of RIP2 in vitro, a large amount of purified protein is required. For this purpose, the full length of RIP2 was cloned from human Ramos (human Burkitt lymphoma) tumor cells and inserted in a prokaryotic expression vector pET22b, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The expression of RIP2 was induced with IPTG. SDS-PAGE analysis showed that recombinant human RIP2 (rhRIP2) was mainly expressed as soluble fraction in the supernatant of the cell lysate. The recombinant protein was subsequently purified by His Trap FF crude to a purity of 90 %. MTT assay of the purified rhRIP2 showed its functional diversity in different cell lines, a specific inhibitory effect on MCF7 cells, but a promotion on the proliferation of Ramos cells. Furthermore, we identified that rhRIP2 could suppress activation of canonical NF-κB in MCF7 cells and activate non-canonical NF-κB signaling in Ramos cells, these data suggested that RIP2 participates in different signaling pathways contributing to its specific effects in vitro. Our results provided new clues to further explore the regulation mechanisms of RIP2 in tumorigenesis.

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Acknowledgments

This research was supported by Grants from the Major State Basic Research Development Program of China (973 Program) (No. 2011CB503803) and National Natural Science Foundation of China (No. 30873030, No. 81071928 and No. 81001175).

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Authors and Affiliations

  1. Laboratory of Genome Engineering, Beijing Institute of Basic Medical Sciences, Beijing, 100850, China

    Xin Cai, Min Wang, Ye Liu, Wengrong Xia, Minji Zou, Jiaxi Wang & Donggang Xu

  2. Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, China

    Haibo Kong

  3. Affiliated Hospital of Military Medical Science Academy, Beijing, 100071, China

    Jing Liu & Hang Su

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  1. Xin Cai
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  2. Min Wang
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  3. Haibo Kong
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  5. Ye Liu
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  6. Wengrong Xia
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  7. Minji Zou
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  8. Jiaxi Wang
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  9. Hang Su
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  10. Donggang Xu
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Correspondence to Hang Su or Donggang Xu.

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D. Xu and S. Hang are co-corresponding authors.

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Cai, X., Wang, M., Kong, H. et al. Prokaryotic expression, purification and functional characterization of recombinant human RIP2. Mol Biol Rep 40, 59–65 (2013). https://doi.org/10.1007/s11033-012-1995-z

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  • DOI: https://doi.org/10.1007/s11033-012-1995-z

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