Expression, Purification And Characterization Of The Extracellular Domain Of Human P-selectin

Selectin family is a kind of adhesion molecules. It contains three numbers, which are E-, P- and L-selectins. They are expressed cell surface of different tissue, and play different roles. P-selectin is an important factor that mediate inflammatory cells migrating to lesion area during the early stage of acute inflammation. P-selectin mediates adhesion of neutrophilic leukocyte, monocytes with platelet and endothelial cells. It is close with the immunologic injury, inflammation, thrombosis and cancer metastasis.The gene of P-selectin locates in 1 chromosome, exhibiting high homogeneity among different species. P-selectin belongs to I-type transmembrane glycoprotein and consists of 789 amino acids. It is shored in theа-granules of platelet and Weibel-Palade of endothelial cell. Its molecular weight is 140kD. P-selectin is made up of five domains from N-terminal to C-terminal, which are calcium dependent lectin domain also called carbohydrate recognition domain , epidermal growth factor-like domain ,a short consensus repeats also called complement regulatory protein domain , transmembrane domain and cytoplasmic tail domain. Among them, the lectin domain and the epidermal factor-like domain exhibit strong relationship with P-selectin fuction.P-selectin primary ligand is P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric molecule rich in O- and N-glycans. PSGL-1 is found on a number of haemopoietic cells such as neutrophils, lymphocytes, eosinophils, monocytes and other myeloid progenitor cells where it mediates tethering and adhesion. Its structure and functions have now been described in detail. Numerous in vivo and in vitro experiments in mice and humans have clearly illustrated the role of P-selectin (and other adhesion molecules) and PSGL-1in supporting platelet-leukocyte interactions and in leukocyte rolling on the endothelium. This may include signaling events within the endothelium.ObjectiveIn order to study the biology function of P-selectin in vitro, we have constructed a chimeric gene of the extracellular domain of the P-selectin fused to the c-Myc domain of mouse. The chimeric gene named extracellular gene was inserted in vector pcDNA3.1, then chimeric protein is expressed in 293T cells, purified the secreted chimeric protein by using Ni-Hisbind. We testified if chimeric protein can interact with HL-60 cells through adhesion experiment.MethodsWe amplified extracellular domain of P-selectin from the vector of P-selectin/IgG with PCR. The amplified extracellular domain gene was inserted in pMD-18T clone vector and identified sequence after testifing with AatII, HindⅢdouble enzyme cut. The right extracellular domain gene was inserted in pX vector (pX Vector contain secreted signal sequence in upriver position). We obtained chimeric extracellular domain gene from pX vector with double enzyme cut. The right chimeric extracellular domain gene was inserted in pcDNA3.1 expression vector and obtained recombinant expression vector pcDNA3.1.Then we have the Constructed recombinant expression vector pcDNA3.1 transformed into DH5αbacterium. After obtaining the highly-purified plasmid, we transfected plasmids into 293T cells. Expression product was detected with ELISA. We make sure that chimeric protein of P-selectin was expressed after being trnsfected 24h and increased at 48h,72h constantly. Abundant chimeric protein was produced from the supernatant of culture media and was purified with Ni-Hisbind affinity Chromatography column and identified with SDS-PAGE and western-blot. At last we tested the activity and adhesion ability to HL-60 cells with adhesion experiment in vitro.Results:We identified the final recombinant expression vector pcDNA3.1 with PCR and NotⅠ, HindⅢdouble enzyme cut, the agarose electrophoresis result showed that there was a specificity band at 720bp place and corresponding to what we anticipated. We delivered the recombinant expression vector to sequence at the Takara Company and the result was absolutely right. After transfecting therecombinant expression vector pcDNA3.1 for 24h,we could test the expression of protein, and continued culturing about 120h, then collected the supernatant of culture media. Protein was purified with Ni-Hisbind affinity Chromatography column, and the SDS-PAGE result showed that there was a specificity band at 40kD place and corresponding to what we calculated. The western-blot result shows that the chimeric protein can specially bind with the c-Myc antibody. The adhesion experiment in vitro showed that the adhesion ability of this chimeric protein with HL-60 cells was remarkably stronger than with control cells. (P<0.05 Compares with the control group has remarkable statistics significance)ConclusionsWe have successfully constructed the recombinant expression vector pcDNA3.1 with extracellular domain, and it can be expressed in the 293T cells by using the transfection of lipid the purified P-selectin chimeric protein of human has biology activity and can specially bind with the c-Myc antibody.