Prokaryotic Expression,Purification,Identification And Interaction Of Human PD-L1 And DCTN4 Genes

In tumor immunotherapy,programmed death ligand-1(PD-L1)is a key protein in immune checkpoints PD-1/PD-L1.In order to inhibit the immune escape mediated by the signaling axis,more and more monoclonal antibodies targeting the PD-1/PD-L1 signal axis have been used in cancer treatment and have achieved certain results.However,due to the limitations of PD-1/PD-L1 monoclonal antibodies,including different cancers' sensitivity to drugs,difficulty in storage and transportation,and high prices,small molecules inhibitors based on the PD-1/PD-L1 signal axis bring new "blood".However,PD-L1 related mechanism including its expression mechanism is not yet fully clear,which is not conducive to its further clinical application.Exploring the interacting protein with PD-L1,clarifying the binding site and interaction mechanism,will provide more information for seeking small molecular inhibitors targeting PD-L1.Our research team used phage display technology to screen the polypeptide D4 that can bind to PD-L1,and searched the database according to the amino acid sequence of the polypeptide,and obtained dynactin subunit 4(DCTN4)that matches the amino acid sequence.The results of Co-IP after overexpressing exogenous PD-L1 and DCTN4 showed that PD-L1 and DCTN4 formed a complex in the HEK293 T cell,and DCTN4 had at least two domains involved in the formation of the complex.In my work,to explore whether PD-L1 and DCTN4 need intermediate auxiliary proteins to form a complex,PD-L1,DCTN4 and truncated proteins were expressed in prokaryotes to study whether there is a direct binding,and to search for the interaction domains.In this study,PD-L1,DCTN4 and their truncated proteins were constructed in p ET28a-TEV plasmids,the E.coli BL21 expression system.First,optimize the PD-L1,DCTN4 and truncated protein expression and purification system.After obtaining PD-L1,DCTN4 and the truncated protein,immunocoprecipitation(Co-IP)was used to capture the complex formed between PD-L1,DCTN4 and the truncated body to find the interaction domains.Using isothermal titration calorimetry(ITC),the interaction between the extracellular region of PD-L1 and DCTN4(1-342aa)was studied,and the strength of the interaction was quantified.The experimental results prove that PD-L1 and DCTN4 have multiple binding sites directly involved in the formation of the complex,which is consistent with the results of Co-IP in vivo.This experiment looks for direct binding sites on the basis of PD-L1 interacting proteins,and provides a new direction for the design of small-molecule peptide drugs targeting PD-L1.