Expression, Purification And Bioactivity Identification Of TACI-Fc And BCMA-Fc Fusion Proteins

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) and B cell maturation antigen (BCMA), which along with BAFF-R, are the three receptors of BLyS. In this project, we cloned the full length of TACI and ligated the extracellular fragment soluble TACI (sTACI) with human IgG1 Fc fragment, and constructed the prokaryotic expression vectors. Then pET28a-TACI-Fc and pET28a-BCMA-Fc recombinant plasmids were induced by IPTG to express the corresponding proteins. Next, the proteins were purified by Protein A chromatography, and their bioactivity was identified by MTT. We provide experimental base for further study on the action mode of TACI/BCMA with BLyS.In summary, the results of this study are as follows:(1)Clone the full length of TACI. The 900bp cDNA was amplified by RT-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 pET28a. The prokaryotic expression plasmid pET28a-TACI-Fc was successfully constructed.(3)Recombinant plasmids pET28a-TACI-Fc and pET28a-BCMA-Fc constructed previously were transformed into E.coli BL21 (DE3) respectively. The expression was induced by IPTG and optimal conditions of the induction were achieved. SDS-PAGE analysis showed that about 40kDa and 35kDa fusion proteins were highly expressed. The expressed proteins were identified by Western Blotting and ELISA. The expressed proteins were purified by Protein A chromatography. SDS-PAGE and Western Blotting analysis showed that the target proteins were highly purified.(4)The bioactivity of the purified proteins was indentified by MTT. The fusion proteins could inhibit the proliferation of BLyS to Nalm-6 cell lines. And the inhibitory effect of TACI-Fc fusion protein was more potent than that of BCMA-Fc.