A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative.
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.
Liu
Haifeng
H
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 200237, Shanghai, China.
Zhou
Xiangshan
X
Zhang
Yuanxing
Y
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*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator