Expression Of Soluble Human PD-1 Extracellular Domain Protein In The Late Logarithmic Phase Of E.coli

BackgroundProgrammed cell death receptor-1(PD-1)is a member of the CD28 immunogloblin superfamily.PD-1 is a type ? transmembrane glycoprotein mainly expressed on the surface of activated T cells,B cells,macrophages and dendritic cells.PD-1 has two cell surface ligands,PD-L1(B7-H1)and PD-L2(B7-CD),which are both members of the B7 superfamily.Among them,PD-L1 is widely expressed on the surface of activated immune cells(such as T and B cells),parenchymal organs,and tumor cells.The expression of PD-L2 is more limited,mainly on the surface of some macrophages and dendritic cells.The interaction between PD-1 and its ligand can provide inhibitory signals which inhibit T cell activation and proliferation,and induce T cell apoptosisThe PD-1/PD-L signaling pathway has a variety of biological functions.The main role is to reduce the immune response of the body against foreign antigens by inhibiting the early activation of T cells.PD-1/PD-L signaling pathway can participate in the regulation of Treg cell differentiation and inhibit the function of effector T cells.It also protects the host in the allograft-versus-host response.In addition,the PD-1/PD-L signaling pathway also plays a key role in tumor immune escape and the pathogenesis of autoimmune diseases.A large number of biologically functional human PD-1 extracellar domain proteins need to be prepared in vitro to further explore the PD-1/PD-L interaction mechanism.At present,most human PD-1 extracellular domain proteins are prepared by eukaryotic expression systems,but their preparation cost is high.The prokaryotic expression system has the advantages of simple operation,short expression cycle,high yield and low production cost.So many researchers choose prokaryotic expression to prepare human PD-1 extracellar domain protein.However,the human PD-1 extracellular domain protein expressed by the E.coli expression system is an inclusion body protein and does not have biological functions.The inclusion body protein must uNdergo a complicated and time-consuming renaturation process.Thus far there is no report that the soluble human PD-1 extracellular domain protein was expressed by the E.coli expression systemIn order to express soluble human PD-1 extracellular domain protein in E.coli,this study first used conventional E.coli expression strategies:common cytoplasmic expression,periplasmic expression,molecular chaperone co-expression,and periplasmic expression and molecular chaperone co-expression;finally,by changing the composition of culture medium and the OD value of the bacterial solution during induction,a method for expressing the soluble human PD-1 extracellular domain protein was established,which is helpful to carry out PD-1 functional studies.PurposeThe soluble human PD-1 extracellular domain protein was expressed by E.coli.Methods1.The successfully constructed pET-28a-PD-1 plasmid was transformed into the BL21(DE3)expression host strain for prokaryotic expression.The induced expression conditions were 37?,220rmp,6h and 16?,220rmp,24h.The OD of the bacterial solution was about 0.6.The final IPTG concentration gradient was set to 0.01mM,0.1mM,0.5 mM,1mM,and 2mM.2.The successfully constructed periplasmic pET-28a-PD-1 plasmid was transformed into the BL21(DE3)expression host strain for prokaryotic expression.The induced expression conditions were 37?,220rmp,6h and 20?,160rmp,24h.The OD of the bacterial solution was about 0.6,and the final IPTG concentration gradient was OmM,0.01 mM,0.5 mM,1 mM,and 2 mM.3.The successfully constructed the pET-32a-Ervl-PDI co-expression plasmid was transformed into BL21(DE3)expression host strains.Six monoclonal strains were selected for prokaryotic expression verification.The induced expression conditions were 37 ?,220rmp,6h and 20 ?,160rmp,24h.The OD of the bacterial solution was about 0.6.The final IPTG concentration was 0.1mM,and the negative control was BL21(DE3)expression host bacteria.4.The successfully constructed pET-32a-Ervl-PDI co-expression plasmid and pET-28a-PD-1 plasmid were transferedinto BL21(DE3)expression host strain for prokaryotic expression.There were two conditions for inducing expression:one is 37?,220nnp,6h,the OD of the bacterial solution was about 0.6,the final IPTG concentration gradient was 0.1mM,0.5mM,1mM,2mM;the other is 20?,160rmp,24h,the OD of the bacterial solution was about 0.6,the final IPTG concentration gradient was 0 mM,0.01 mM,0.1 mM,1 mM,2 mM,and the negative control was pET-28a empty vector.5.The successfully constructed periplasmic pET-28a-PD-1 plasmid and pET-32a-Ervl-PDI co-expression plasmid were transfered into the BL21(DE3)expression host strain for prokaryotic expression.The induced expression conditions were 20?,160rmp,24 hours.The OD value of the bacterial solution was about 0.6,and the final IPTG concentration gradient was 0 mh,0.01 mM,0.1 mM,1 mM,and 2 mM.6.The pET-28a-PD-1 plasmid was transformed into the BL21(DE3)expression host strain for prokaryotic expression.The OD value of the bacterial solution was about 2.0 when the IPTG was added.The medium for inducing expression was changed from LB medium to XYB medium.The human PD-1 extracellular domain protein was expressed at 16?,160 rpm,1 mM IPTG and was cultured for 12-16 h.7.The human PD-1 extracellular domain protein was purified by two steps of nickel column and ion exchange chromatography,and was quantified by BCA method.8.The activity of human PD-1 extracellular domain protein was detected by competitive ELISA.ResultOne L E.coli obtained 97 ?g of soluble human PD-1 extracellular domain protein with binding activity.ConclusionThis study established an effective and simple method suitable for the expression of soluble human PD-1 extracellular domain protein in the E.coli expression system,which is helpful for its subsequent functional research.