文献类型：期刊文献

英文题名：A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative

作者：Liu, Haifeng[1,2]; Zhou, Xiangshan[1]; Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shandong Dongeejiao Grp, Shandong 252201, Peoples R China

年份：2006

卷号：28

期号：7

起止页码：457

外文期刊名：BIOTECHNOLOGY LETTERS

收录：;EI(收录号：2006179839761);WOS:【SCI-EXPANDED(收录号:WOS:000236846500001)】；

语种：英文

外文关键词：inclusion body; on-column refolding; size-exclusion chromatography; tissue-type plasminogen activator derivative

摘要：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.