title: A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative.
abstract: 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 200237, Shanghai, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Liu
authors.forename: Xiangshan
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D006801:Humans; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator