Expression And Purification Of GST-hDSL Fusion Protein In Prokaryotic Cells And Preparation Of Its Chicken Egg Yolk Antibody

Notch signal pathway is widely recognized as a key pathway, which plays a key role in regulating cell differentiation and determines the cell fate. It consists of Notch ligand、Notch receptor and downstream signaling molecules. Recent studies have discovered that Notch receptor is expressed on the cell surface of hematopoietic stem/progenitor cells and mature blood cells while the Notch ligand in the surface of hematopoietic matrix cells, which indicated that Notch pathway may play a significant role in regulating production and development of the hematopoietic system. In the culture of stem cell in vitro, the activated Notch pathway instructed the stem cell to renew itself. Hence, the activated Notch pathway may promote the expansion of the hematopoietic stem/progenitor cells in vitro.Notch ligands exist as both transmembrane and secreted proteins. The DSL domain,located in the extracellular area, is a highly-conserved domain of all ligands. It is an essential structure for the interaction between the ligand and the receptor, and the activation of Notch signaling pathway. hDelta-like-1(hDll-1)is one of the human Notch ligand. We have selected the mini function protein domain of the hDll1 by analyzing the protein structures, which includes the DSL domain and the N-terminal 50 amino acids, called hDll-1NDSL (hDSL in short). In order to study the role of hDll-1 in the expansion of umbilical cord blood hematopoietic stem/progenitor cells in vitro, we cloned the hDSL into prokaryotic expression vector pGEX-2T,then transformed into E. coli DH5α. Fusion protein GST-hDSL was expressed in E. coli via IPTG induction. The expressed proteins were purified using DEAE anion exchange column. The purified GST-hDSL was further mixed with Freund’s complete and incomplete adjuvant and used to immunize egg-laying hens. The specificity of the antibody was identified by Western blot, which will be useful to study the role of GST-hDSL in the future.The hDSL gene was cloned by PCR from recombinant plasmid pBlue-hDLL-1, and was shown as 300bp fragment in size by agarose gel electrophoresis. Recombinant plasmid was successfully constructed and identified using bacteria colony PCR and DNA sequencing. Recombinant GST-hDSL protein was successfully expressed in DH5αand the expressed protein in E. coli mainly existed in the inclusion bodies. The molecuLar weight of GST-hDSL is 37 kDa by SDS-PAGE analysis. The optimized induction conditions for the expression of the protein included incubation at 37℃f or 4h with 1mM IPTG and 100μg/mL ampicilin. GST-hDSL was expressed in large-scale under above induction conditions. 0.5g of inclusion bodies was collected after destruction of bacteria from 1000 mL induced culture. 50mL of target protein solution with 0.8mg/mL protein concentration was obtained after DEAE anion exchange chromatography. The anti-GST-hDSL antibody (IgY) was collected through immunized egg yolk and identified by Western-blot.In conclusion, the sequence of GST-hDSL was cloned by PCR from plasmid pBlue-hDLL-1 and identified by sequence analysis. GST-hDSL recombinant protein was expressed in E. coli inclusion bodies. Anti-GST-hDSL polyclonal antibody was produced, and detected the purified GST-hDSL recombinant protein.