Purification and Efficient Refolding Process for Recombinant Tissue-Type Plasminogen Activator Derivative (Reteplase) Using Glycerol and Tranexamic Acid Publisher



Mousavi SB¹ ; Fazeli A³ ; Shojaosadati SA¹ ; Fazeli MR² ; Hasheminajafabadi S¹ Show Affiliations
Source: Process Biochemistry Published:2017

Abstract

In this study, a novel and economic method for refolding and purifying recombinant tissue plasminogen activator derivative (r-PA; reteplase) was developed. Reteplase with nine disulfide bonds in its complex structure is expressed in the form of inclusion bodies in Escherichia coli and requires tedious dissolving and refolding processes to achieve its biological activity. Among the different refolding additives that were evaluated, glycerol and tranexamic acid (Txa) were found to be more effective in increasing the refolding yield of reteplase. Using response surface methodology, a solution containing 3.5 M urea, 33% (v/v) glycerol, and 400 mM Txa was found to give the highest refolding yield. The synergic effect of urea, glycerol, and Txa under optimum conditions for a reteplase concentration of 25 μg ml−1 resulted in a high refolding yield of 76.41%. Increased reteplase concentration in the refolding buffer was achieved using the pulse-fed method. In the pulse-fed method, a refolding yield of 49.53% was achieved for a final reteplase concentration of 300 μg ml−1. Using Txa as a novel refolding aid for reteplase instead of ionic amino acids like L-Arginine allowed to purify the refolded reteplase directly by cation-exchange chromatography with high purity. © 2016 Elsevier Ltd