Expression, Purification And Preliminary Indentification Of HDll1^ext-Fc Fusion Protein In CHO-S Cells

The first notch gene was found in Drosophila, it was named that due to the absence of this gene in Drosophila wing edges can cause a gap (notch). Notch signaling pathway is widely present in the spine and non-vertebrates, it is one of the important paths to determine cell fate. Notch receptors are heterodimers consisting of the extracellular region (extracellular Notch, ECN) and transmembrane region/intracellular domain (Notch transmembrane, NTM) composed of two subunits, the two are covalently bonded together and having Ca2+-dependent. There are two kinds of Drosophila Notch ligands, Delta and Serrate.Nematodes Notch ligands with Lag2. Mammals have multiple Notch ligands, and highly similar ligand Delta called Delta or Delta-like (Delta-like, Dll), and Serrate similar called Serrate or Jagged. Recently,We totally found five species notch ligands in the human body, namely Delta-like-1, Delta-like-3, Delta-like-4, Jaggedl, Jagged2. As an important human Notch ligand Delta-like-1,it is a single transmembrane protein consisting of723amino acids.there are539amino acids in the extracellular region which have important biological activity contains an N-terminal conserved DSL (Delta/Serrate/Lag2) motif, and eight epidermal growth factor-like repeats (epidermal growth factor-like repeats, EGF-R) domains. The DSL domain of Notch receptor binding, receptor heterodimers promote separation, and then activate downstream signaling receptor, after shearing three times, eventually releasing intracellular domain(NICD) to the nucleus.Finally the NICD domain activates hair like split related enhancer (hairy enhancer of transcription split, Hes) and other target genes,thereby regulated cell differentiation and tissue. Intracellular domain function is not yet clear, studies have shown that intracellular of Deltal may induce cell growth inhibition.Recent years, studies have shown that Notch signaling pathway was associated with oncogenesis, such as Purow BW et al found that Notch1and Notch4ligand Delta-like-1, Jaggedl is overexpressed in a variety of glial cell tumor (2005), and in the United the case of other cytokines, the extracellular region of hDlllcan inhibit the differentiation of hematopoietic stem cells and promote their proliferation. Lu thrive, such as human cloning and expression of the Notch ligand Delta-like-1(hDll1) extracellular region, and the colony culture tests confirmed its original amplification of hematopoietic progenitor cells.In the end, according to a known sequence, hDlllext synthetic gene fragment was amplified by PCR, Nhel and BamHl ligated to pIRES2-EGFP-Fc, and transform the liagated product into competent cells, applied to the anti-containing kana the screening of LB solid medium, inverted overnight, positive clones were screened by colony PCR to verify the correct sequences, the correct recombinant plasmid was transfected into CHO-S cells, the high-expressing cell lines were selected by G418, and then the rProtein A affinity column was used to purify protein, and through both the expression of downstream of Notch signaling molecule Hesl and dual luciferase reporter gene assay to detect the activity of recombinant protein.Finally,we obtained highly expressed hDll1ext-Fc of CHO-S cell line10C7, by WAVE culture the density was up to6.27x106cells/mL, nearly two weeks after culture, the cells almost died, collected the supernatant about1.1L, the protein concentration was47.8μg/mL in supernatant, total protein was estimated to52.58mg. The above supernatant was purified to obtain a higher purity fusion protein, protein concentration was measured at1.5mg/mL, a volume of30mL, total protein was45mg, rProtein A can be obtained affinity column recoveries45mg/52.58mg=85.6%. With bioassay experiments showed that soluble hDll1ext-Fc can also activate Hesl reporter gene, and can increase the expression of Notch downstream molecule Hesl to prove that it can activate the Notch signaling pathway.High purity purification obtained by the experiment with biological activity hDll1ext-Fc fusion protein, laid an important foundation for further study in its function.