Two observed regions in B lymphocyte stimulator important for its biological activity

B lymphocyte stimulator (BLyS),a member of the tumor necrosis factor superfamily of ligands,is a crucial survival factor for B cells.We successfully constructed seven mutants of the functional soluble fragment of human BLyS (named cBLyS,amino acid 134-285),including three deletion mutants and four site-directed mutants.All the mutant proteins were expressed in Escherichia coli and purified by Ni- NTA affinity chromatography.The biological activities of these mutants were assessed by the ligand-recep- tor binding assay,B cell proliferation assay and immune effect response in vivo.Our results indicated that four residues,H~(218),F~(220),T~(228) and L~(229),are indispensable for the biological activity of cBLyS,whereas two regions,amino acid 134-148 and amino acid 271-285,are related to the biological activity of BLyS.The protein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgM titer.The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS.This indicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.