Secretory expression of negative regulatory region of human Notch1 in Escherichia coli and preparation of a functional polyclonal antibody.
Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.
['Zhang|Dengyang|D|http://orcid.org/0000-0001-7844-1779', 'Sun|Tianzhu|T|', 'Yan|Jingyun|J|', 'Wang|Xuanjun|X|', 'Sheng|Jun|J|']
D000906:Antibodies D000937:Antigen-Antibody Reactions D000941:Antigens D003593:Cytoplasm D004926:Escherichia coli D057809:HEK293 Cells D006367:HeLa Cells D006801:Humans D012333:RNA, Messenger D051881:Receptor, Notch1 D011994:Recombinant Proteins
negative regulatory region immunization Notch1 protein low-phosphate induced expression signal transduction soluble expression