Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution

• Mild solubilization of aggregated VAS-TRAIL was achieved by a triple-denature solution consisting of urea, l-arginine, and alkaline PH.
• VAS-TRAIL solubilized by the triple-denature solution resulted in an efficient recovery of soluble protein over 50%.
• Refolded VAS-TRAIL fusion protein exhibited apoptotic activities toward tumor and endothelial cells.

VAS-TRAIL is a bifunctional fusion protein that combines anti-angiogenic activity with tumor-selective apoptotic activity for enhanced anti-tumor efficacy. VAS-TRAIL is expressed as inclusion body in Escherichia coli, but protein refolding is difficult to achieve and results in low yields of bioactive protein. In this study, we describe an efficient method for VAS-TRAIL refolding. The solubilization of aggregated VAS-TRAIL was achieved by a triple agent solution, which consists of an alkaline solution (pH 11.5) containing 0.4 M l-arginine and 2 M urea. The solubilized protein showed high purity and preserved secondary structure according to fluorescence properties. VAS-TRAIL refolding was performed through stepwise dialysis and resulted in more than 50% recovery of the soluble protein. The function of l-arginine was additive with alkaline pH, as shown by the significant improvement in refolding yield (≈30%) by l-arginine-containing solubilization solutions compared with alkaline solubilization solutions without l-arginine. The refolded VAS-TRAIL also showed β-sheet structures and the propensity for oligomerization. Bioassays showed that the refolded fusion protein exhibited the expected activities, including its apoptotic activities toward tumor and endothelial cells, which proposed its promising therapeutic potential.