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.

Structural Biology and Protein Engineering of Thrombolytics

“…Production in yeasts generally leads to higher yields and absence of endotoxin when compared to bacteria [[363], [364], [365]]. Other avenues are auto-induction, growth medium or cultivation protocol optimization [366,367], development of new refolding protocols [[368], [369], [370]] or new mammalian cell lines [371]. To avoid the demanding work with mammalian cell cultures, eukaryotic plasminogen activators can be produced in plants or extracted from the milk of transgenic animals [[372], [373], [374], [375], [376]].…”

Urea-Gradient Protein Refolding in Size Exclusion Chromatography

“…The total enzyme activity and specific activity of recombinant carboxypeptidase B refolded by UGSEC were 45% and 17% higher than those by dilution refolding. Recombinant human tissue-type plasminogen activator derivative (r-PA) fused with thioredoxin (Trx) expressed in E. coli as inclusion bodies was refolded by UGSEC [12]. Using a Sephacryl S-200 gel column and a urea gradient from 8 M to 2 M in 15 mL, the specific activity and activity recovery increased by 91% and 96% when compared with normal SEC.…”