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Development of a Two-Step Refolding Method for Reteplase, a Rich Disulfide-Bonded Protein Publisher



Mousavi SB1 ; Fazeli A2 ; Shojaosadati SA1 ; Fazeli MR3, 4
Authors
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Authors Affiliations
  1. 1. Biotechnology Group, Chemical Engineering Faculty, Tarbiat Modares University, P.O. Box 14115-114, Tehran, Iran
  2. 2. The institute of Pharmaceutical Sciences, Tehran University of Medical Sciences, Tehran, Iran
  3. 3. Pharmaceutical Quality Assurance Research Center, The institute of Pharmaceutical Sciences, Tehran University of Medical Sciences, Tehran, Iran
  4. 4. Department of Drug & Food Control, Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Source: Process Biochemistry Published:2018


Abstract

Correct formation of disulfide bonds is crucial in the refolding of multi-disulfide-bonded inclusion body proteins. Covalent aggregation of proteins due to incorrect cross-linking of disulfide bonds decreases refolding yield. This study presents a two-step refolding method for increasing refolding yield of disulfide-bonded proteins. In this method disulfide bond formation is prevented in the first step of refolding, then in second step disulfide exchange reactions are promoted by gradual increasing of pH and cysteine concentration in refolding buffer. Recombinant tissue plasminogen activator (r-TPA, reteplase) with nine disulfide bonds was used as the model protein. Refolding yield of reteplase with two-step refolding method improved by 2.1-fold compared to dilution refolding. The successful application of two-step strategy in improving refolding yield of reteplase reveals the efficacy of this strategy for decreasing aggregation and improving correct refolding of inclusion body proteins. © 2018 Elsevier Ltd
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Mohammad Reza Fazeli (3)
Elham Kazemirad (1)