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.
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Revisions requested 31 October 2005; Revisions received 20 December 2005
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Liu, H., Zhou, X. & Zhang, Y. A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative. Biotechnol Lett 28, 457–463 (2006). https://doi.org/10.1007/s10529-006-0001-z
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DOI: https://doi.org/10.1007/s10529-006-0001-z