Optimizing refolding condition for recombinant tissue plasminogen activator

Document Type: Research Paper

Authors

1 Infectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65813, Tabriz, I.R. Iran.

2 Biotechnology Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-6581,Tabriz, I.R. Iran.

3 Drug Applied Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, I.R.Iran.

4 Biotechnology Research Center, Pasteur Institute of Iran, P.O. Box 13164, Tehran, I.R.Iran.

5 Drug Applied Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, I.R. Iran.

Abstract

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture
medium and in vitro refolding to increase recombinant proteins production. Additives increase protein
refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process.
In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA)
in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA
inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution
method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and
Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM.
Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presenceof higher concentration of L-arginine (> 500 mM) or pH (>10) is not associated with higher activity.

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