Diao Zhenyu, Ye Tingmei, Cao Peng, Zhang Jing, Mei Jingjing, Lin Zhihua, Zhang Shuangquan
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, Jiangsu, PR China.
Protein Expr Purif. 2007 Jul;54(1):11-7. doi: 10.1016/j.pep.2007.01.002. Epub 2007 Jan 9.
The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.
B淋巴细胞刺激因子（BAFF）是肿瘤坏死因子（TNF）配体家族的一个新成员，在B淋巴细胞的成熟和存活中起重要作用。在此，编码BAFF细胞外结构域（hsBAFF）的cDNA已被克隆到分泌表达宿主毕赤酵母中。对甲醇诱导表达菌株的培养液进行SDS-PAGE和蛋白质印迹分析表明，重组hsBAFF（一种20.2 kDa的糖基化蛋白）被分泌到培养基中。使用DEAE-琼脂糖离子交换和Superdex 75尺寸排阻色谱步骤将重组蛋白纯化至纯度大于95%。最后，从1 L上清液中获得了102 mg高纯度的该蛋白，通过N端氨基酸序列分析证实了其与hsBAFF的一致性。重组hsBAFF的生物活性通过该蛋白在体外刺激人B淋巴细胞增殖的能力得到证实。我们的结果表明，毕赤酵母表达系统可用于大量生产用于研究和工业目的的具有完全功能的hsBAFF。
