功能性表达II型跨膜蛋白胞外区与Fc区的融合蛋白.docVIP

 Functional expression of the Fc-fused extracellular domains 5 10 15 20 25 30 35 40 45 of group II membrane proteins# Abstract: The IgG Fc moiety is widely used as an affinity tag that confers an intrinsic dimerization and provides the recombinant proteins with the bivalent binding function to increase the binding avidity. However, the complicated delivery mechanism of group II membrane proteins makes it difficult to decide the fusion pattern of their extracellular domains (ECDs) with Fc moiety. In this study, we compared the expression of three ECDs of group II membrane proteins including CLEC-2, Dectin-1, and LOX-1 by fusion of Fc moiety from different species and at different locations. We found that these fusion patterns of the ECDs with Fc moiety had different effects on their glycosylation modifications and structural conformations. The pattern of ECD-Fc fusion order produced the functionally active recombinant proteins with native conformation while the pattern of Fc-ECD fusion order led to the altered glycosylation and the improper conformation which abolished the binding of these proteins with their ligands. Meanwhile, our results indicated that the secretion of mouse Fc (mFc)-fused ECD of CLEC-2 was more efficient than that of rabbit Fc (rFc)-fused ECD of CLEC-2, while rFc moiety was more sensitive for detection compared with mFc moiety. Altogether, we provide a favorable fusion pattern of Fc moiety with the ECDs of group II transmembrane proteins and simultaneously present an alternative selection of mFc and rFc for different aims. Keywords: Fc moiety; extracellular domain; group II membrane proteins; CLEC-2 0 Introduction There are a number of transmembrane proteins expressed on the cell surface. Generally, these transmembrane proteins can be divided into three groups based on their orientation in the membrane[1, 2]. Group I proteins have a single transmembrane region with the C-terminal at the cytoplasmic side of the membrane. Group II proteins h