Analyzing Conformation of Fusion Protein on Bivalent Single-Chain Antibody with (Gly4Ser)n

scFv (single-chain Fv fragment) is the gene engineered antibody employed widely in at present. Recently there have been some progress on colorectal cancer diagnosis and treatment-oriented with scFv as carrier, but it is differential on affinity and anti-tumor activity of scFv with that of derived factor. Proper linker could make scFv have more suitable biological activity on clinical application. So it becomes a key step that how to choose and design a proper linker. In this work, the (Gly₄Ser)n is used as the linker to connect VH and VL separately (n=0,1,2,3,4,5,6,7,8,9). and the 3D structure of corresponding scFv is reconstructed by homology modeling. The End-End distance of scFy protein with different linker (G₄S)n, which 3D structure coordinate system are normalized, are compared to evaluate the effect of linker length to VH and VL. In molecular biology experiment, ND-1scFv-n are expressed and analyzed with n=1,3,5,7,9, their biologic activity active are consistent with what estimated by End-End distance. So the index to estimate biologic activity of scFv with its End-End distance is presented and could be helpful to further understand the relationship between the spatial structure of single-chain antibody and the physical and chemical properties.