Computer Simulation and Additive-Based Refolding Process of Cysteine-Rich Proteins: VEGF-A as a Model

Abstract

Refolding of cysteine-rich protein for establishing native conformation and a biologically active form is the most challenging step in recombinant protein synthesis. In this study, expressed vascular endothelial growth factor-A (VEGF-A), as a cysteine-rich protein, in a prokaryotic expression cell was refolded based on computer simulation technique and multiple chemical additive-based buffers to recover its biologically active form. For this purpose, cloned and expressed VEGF-A in Escherichia coli BL21 (DE3) was purified and dialyzed by a basic buffer containing nine diverse chemical additives. In parallel with the evaluations of the applied additives, professional computer simulation software was also used. The activity of refolded protein was evaluated in differentiation of mesenchymal stem cells (MSCs) to the endothelial cells (ECs). The results showed that dialyzing the produced recombinant VEGF-A in chemical additive-based buffers containing cysteine, 1, 4-dithiothreitol (DTT), arginine, and Triton X-100 led to efficient VEGF-A refolding. The results of flowcytometry analysis indicated that CD31 and CD144 as the specific ECs markers in VEGF-A treated MSCs were 31 and 73%, respectively. Protein refolding method using chemical additive-based buffers containing cysteine, DTT, arginine and Triton X-100 was the best accessible technique for refolding cysteine-rich recombinant VEGF-A.