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-receptor binding assay, B cell proliferation assay and immune effect response in vivo. Our results indicated that four residues, H218, F220, T228 and L229, 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.