New Type B Lymphocyte Blockers (taci) Protein Engineering

B lymphocyte stimulator (BLyS), also known as B cell-activating factor belonged to TNF family (BAFF), is a member of tumor necrosis factor (TNF) family. Transmembrane activator and CAML interactor (TACI) binds to two ligands including APRIL and BAFF with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular regions while the other known high affinity receptors such as BCMA and BR3 binding to the two ligands contain only a single or partial CRD. Human IgG1 Fc fusion proteins with the extracellular domain of TACI (eTACI), also called decoy receptors, have been used as a potential antagonists to block APRIL activities. In order to design a novel APRIL antagonist, computer-aided homologue modeling was used to construct a CRD2-TACI-Fc fusion protein based on the crystal structures of TACI and Fc fragment. A DNA fragment encoding CRD2-TACI-Fc fusion protein was constructed and introduced into Pichia pastoris for expression. Next, the proteins were purified by Protein A chromatography, and their bioactivity was identified by CCK-8.We provide experimental base for further study on the action mode of TACI/BCMA with APRIL.In summary, the results of this study are as follows:(1) Clone the extracellular fragment soluble TACI. The DNA was amplified by PCR, which was consistent with the sequence reported in GenBank.(2)The extracellular fragment soluble TACI (sTACI) was ligated with human IgG1 Fc fragment. sTACI-Fc was subcloned into expression vector GS115. The Pichia pastoris expression plasmid GS115-CRD2-TACI-Fc was successfully constructed.(3) SDS-PAGE analysis showed that about 46kDa fusion proteins were highly expressed. The expressed proteins were identified by Western Blot and MS.The expressed proteins were purified by Protein A chromatography. SDS-PAGE and Western Blot analysis showed that the target proteins were highly purified.(4)The bioactivity of the purified proteins was indentified by CCK-8. The fusion proteins could inhibit the proliferation of APRIL to Raji cell lines. And the inhibitory effect of CRD2-TACI-Fc fusion protein was more potent than that of TACI-Fc.