title: Prokaryotic expression, purification and functional characterization of recombinant human RIP2.
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.
authors.affiliation: Laboratory of Genome Engineering, Beijing Institute of Basic Medical Sciences, Beijing 100850, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Cai
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Haibo
authors.initials: H
authors.lastname: Kong
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Ye
authors.initials: Y
authors.lastname: Liu
authors.forename: Wengrong
authors.initials: W
authors.lastname: Xia
authors.forename: Minji
authors.initials: M
authors.lastname: Zou
authors.forename: Jiaxi
authors.initials: J
authors.lastname: Wang
authors.forename: Hang
authors.initials: H
authors.lastname: Su
authors.forename: Donggang
authors.initials: D
authors.lastname: Xu
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D061986:MCF-7 Cells; D016328:NF-kappa B / Q000378:metabolism; D010957:Plasmids; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D016328:NF-kappa B; D011994:Recombinant Proteins; C506253:RIPK2 protein, human; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2