Guo Q R, Tong W Y, Wei D Z, Tao X Y
Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Prikl Biokhim Mikrobiol. 2008 Mar-Apr;44(2):187-92.
In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.
在本研究中，对重组大肠杆菌BL21(DE3)中融合蛋白硫氧还蛋白(Trx)-可溶性B淋巴细胞刺激因子(sBLyS)在摇瓶和生物反应器中的表达条件进行了优化，该重组大肠杆菌带有编码Trx-sBLyS融合蛋白基因的pET系统，并对sBLyS的纯化方法进行了优化，以有效获得高纯度的具有生物活性的蛋白sBLyS。优化发酵条件后，在30 L生物反应器中获得了约250 mg Trx-sBLyS/g干细胞重(1686 mg Trx-sBLyS/L)的产量，可溶性Trx-sBLyS的表达水平约为38.5%。按照亲和层析、肠激酶酶切和DEAE离子交换层析的顺序完成优化的纯化程序后，每升发酵液可获得约200 mg sBLyS，纯度约为95%，产率接近30%。此外，通过二维凝胶电泳和SDS-PAGE分析测定了纯化后sBLyS的分子量(MW)和等电点(pI)，估计分别超过16 kDa和约pH 4.15。另外，通过两种生物活性分析方法对发酵液中可溶性Trx-sBLyS和纯化后的sBLyS的生物活性进行了测试。良好的发酵产量以及纯度、产率和生物活性均令人满意的纯化sBLyS产品表明，sBLyS的生产工艺在工业上具有广阔的应用前景。
