Liu Haifeng, Zhou Xiangshan, Zhang Yuanxing
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 200237, Shanghai, China.
Biotechnol Lett. 2006 Apr;28(7):457-63. doi: 10.1007/s10529-006-0001-z.
Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
与硫氧还蛋白（Trx）融合的重组人组织型纤溶酶原激活剂衍生物（r-PA）在大肠杆菌中表达。所得融合蛋白Trx-r-PA几乎完全以包涵体形式存在且无活性。研究了不同的复性策略，包括对溶解的Trx-r-PA包涵体进行不同的后处理、使用三种凝胶类型（Sephacryl S-200、S-300和S-400）通过尺寸排阻色谱（SEC）进行柱上复性、用尿素梯度的Sephacryl S-200进行复性以及复性过程中的两阶段温度控制。建立了一种针对Trx-r-PA包涵体的优化柱上复性方法。收集的Trx-r-PA包涵体溶解于6 M盐酸胍（Gdm.HCl）中，变性蛋白通过G25柱与二硫苏糖醇（DTT）和Gdm.HCl分离，并同时溶解于含有氧化型谷胱甘肽（GSSG）的8 M尿素中。最后采用在Sephacryl S-200柱上以递减尿素梯度结合两阶段温度控制对Trx-r-PA蛋白进行复性，与常规稀释复性相比，复性蛋白的活性回收率从3.6%提高到了13.8%。
