| Sialic acid-binding immunoglobulin-like lectin(Siglec)is a type I transmembrane protein specifically bound to sialic acid,which is mainly expressed on the surface of immune cells and participates in the regulation of the transition of immune cell activation and resting state.The recombinant Siglec-Fc protein,fusing the Siglec extracellular domain and antibody Fc domain,is a commonly used Siglec ligand screening tool for figuring out Siglec preference bound glycan structure or sialylated glycoproteins,glycolipids and other ligands.At the same time,as an antibody-like protein,Siglec-Fc protein can specifically bind with sialic acid and participate in the antibodymediated biological processes due to its structural basis(Fc domain).Thus,it has the application prospect of participating in the targeted sialic acid-Siglec interaction in vivo and participating in cancer treatment as a medical protein.Glycosylation is an important protein post-translational modification,which affects the biological function of pharmaceutical glycoproteins,such as corefucosylation modification reducing antibody-dependent cellular cytotoxicity(ADCC).So in industrial production,the glycosylation modification of pharmaceutical glycoproteins such as antibodies has become a key quality control factor.Different protein expression systems have different preference of glycosylation modification,so in the production of antibody proteins,evaluating the influence of the expression system on the characteristics of proteins such as glycosylation modification of the target protein,and selecting the best expression system is the first step of efficient protein production.In order to select the best expression system suitable for Siglec-Fc antibody protein and lay the foundation for the subsequent large-scale production of the protein and in vitro application research,we selected two mammalian cell expression systems,human embryonic kidney(HEK293)and Chinese hamster ovary(CHO),to express Siglec9-Fc protein.Analyzed the Siglec9-Fc protein properites such as glycosylation expressing in these two manmmalian cells and used the produced Siglec9-Fc to do the ligand identification.The main results obtained in this article are as follows:1)We used dot blot to analyze the protein expression yield,using WB to analyze protein dimer structure,using flow cytometry to analyze sialic acid binding activity and mass spectrometry analysis to analyze glycosylation modification characteristics of Siglec9-Fc protein.It was found that the dimerization ratio of the target protein expressed by different systems was the same.The expressed Siglec-Fc proteins all had sialic acid binding activity,and there was no significant difference.Through the optimization of cell culture conditions,it was found that the expression of protein of interest in CHO cells was higher than that of HEK293 cells.The Siglec9-Fc protein expressed by CHO cells had a high sialic acid modification and a lower core-fucosylation modification,which is theoretically more suitable for the production of antibody-like proteins represented by Siglec9-Fc with high ADCC activity and long half-life;2)Using the expressed Siglec9-Fc as the screening protein,the Siglec9 interaction ligand was studied by flow cytometry,immunofluorescence,mass spectrometry and other methods.The content and distribution of Siglec9 ligand on the surface of cancer cells of different tissue sources were analyzed,and the potential proteins with interaction with Siglec9,such as Basigin,integrin β,CD44,LGALS1 and LGALS3 BP,were explored in bladder cancer cells;3)The gene editing technology was used to knock out the α1-6 fucosyltransferase coding gene FUT8 or Fut8 which is associated with core-fucosylation modification in the glycosylation pathway of HEK293 or CHO cells.The glycosylation modification of Siglec9-Fc expressed in gene knock out cells was analyzed by mass spectrometry.And the core-fucosylation modification-deficient Siglec9-Fc protein was obtained. |
PDF Full Text Request