Man B Lymphocyte Stimulator Factor Cloning, Expression, Purification And Activity

Human B cell activating factor of the TNF family (hBAFF) is a co-stimul -ated factor. In this paper, on the one hand, a fusion between gene encoding fl -uoresce enhanced green fluorescent protein variant (EGFP) and soluble domain of hBAFF by the DNA sequence encoding the linker sequence (Gly₄Ser)₂ was constructed and expressed in Escherichia coli. While the fusion protein was analyzed by Western blotting assay, measurement of fluorescence spectra and effe -ct of purifyed fusion protein on B cell proliferation in vitro. On the other han -d, hBAFF was expressed in Pichia pastoris, and product was analyzed by SD S-PAGE, Western blotting and ELISA assays.1 Expression, purification and activities assay of EGFP-hBAFF fusion protein.The fragment encoding EGFP-hBAFF, which had the linker sequence (Gly₄ Ser)₂ between EGFP and hBAFF was obtained by two rounds of PCR. The E GFP-hBAFF was expressed in E.coli BL21 (DE3), and then the protein was pu -rifled by immobilized Ni²⁺-IDA affinity chromatography. The purified fusion p -rotein retained the fluorescence activity of parental EGFP and co-stimulated hu -man B lymphocyte proliferation in vitro.2 Expression and analysis assays of hBAFF in Pichia pastorisThe fragment encoding hBAFF was inserted in frame into pPIC9 expressio -n vector. The constructed recombinant plasmid pPIC9-hBAFF was digested, an -d then the fragment was transferred into Pichia pastoris GS115. After that, se -veral positive transformations were induced by methanol. And the induced con -ditions were optimized and product was analyzed by Western blotting, ELISA assays, and so on. The result showed that the fusion protein had the largest qu -antity when the strains were induced at 20℃ after 72 hours.