Expression of a Siglec-Fc Protein and Its Characterization
Author:
Chi Kaijun1, Xu Huilin1, Li Hanjie1, Yang Ganglong2, Zhou Xiaoman1ORCID, Gao Xiao-Dong12
Affiliation:
1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
Abstract
The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.
Funder
National Natural Science Foundation of China China Postdoctoral Science Foundation
Subject
General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology
Reference40 articles.
1. Varki, A., Cummings, R.D., Esko, J.D., Stanley, P., Hart, G.W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N.H., and Prestegard, J.H. (2022). Essentials of Glycobiology, Cold Spring Harbor Laboratory Press. [4th ed.]. 2. Siglecs as Immune Cell Checkpoints in Disease;Duan;Annu. Rev. Immunol.,2020 3. Glycosylation in cancer: Mechanisms and clinical implications;Pinho;Nat. Rev. Cancer,2015 4. Zhou, X., Yang, G., and Guan, F. (2020). Biological Functions and Analytical Strategies of Sialic Acids in Tumor. Cells, 9. 5. Sialoglycans and Siglecs Can Shape the Tumor Immune Microenvironment;Santegoets;Trends Immunol.,2020
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