title: Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution.
abstract: 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 0.4M l-arginine and 2M 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jiying
authors.initials: J
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Zhanqing
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Liying
authors.initials: L
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: ylshen@ecust.edu.cn.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Shen
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001120:Arginine / Q000737:chemistry; D037282:Calreticulin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D012996:Solutions; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D014508:Urea / Q000737:chemistry
chemical_list: D037282:Calreticulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012996:Solutions; D053221:TNF-Related Apoptosis-Inducing Ligand; C121986:vasostatin; D014508:Urea; D001120:Arginine
keywords: Mild solubilization; Refolding; Triple agent solution; VAS-TRAIL fusion protein; l-Arginine