Document Detail

Prokaryotic expression, purification and functional characterization of recombinant human RIP2.
MedLine Citation:
PMID:  23135732     Owner:  NLM     Status:  Publisher    
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.
Xin Cai; Min Wang; Haibo Kong; Jing Liu; Ye Liu; Wengrong Xia; Minji Zou; Jiaxi Wang; Hang Su; Donggang Xu
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-8
Journal Detail:
Title:  Molecular biology reports     Volume:  -     ISSN:  1573-4978     ISO Abbreviation:  Mol. Biol. Rep.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-8     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0403234     Medline TA:  Mol Biol Rep     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Laboratory of Genome Engineering, Beijing Institute of Basic Medical Sciences, Beijing, 100850, China.
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