Production optimization of recombinant human vascular endothelial growth factor in tobacco (Nicotiana Benthamiana)

Abstract

Molecular pharming represents the innovative development of plants as alternative production platforms for recombinant proteins. Human vascular endothelial growth factor (VEGF) is one of the growth factors that has displayed eminent roles in wound healing. This study aimed to develop optimized expression conditions and rapid production of recombinant VEGF in Nicotiana benthamiana. Herein, four plant expression constructs, particularly pBYR2e-SP-VEGF-His-SEKDEL (S-V-H-K), pBYR2e-SP-VEGF-His (S-V-H), pBYR2e-SP-His-VEGF-SEKDEL (S-H-V-K) and pBYR2e-SP-His-VEGF (S-H-V), were utilized and inserted into geminiviral vector pBYR2e-K2Md for transient expression in N. benthamiana. The expression level of all the four constructs was examined and the optimal harvest time for high protein expression was identified. High level of plant-produced VEGF protein expression in both S-V-H-K and S-V-H constructs were apparently detected on day 3 post-infiltration. The recombinant protein was then purified from the crude extracts by using nickel affinity column chromatography and confirmed by Western blot probed with anti-His and anti-VEGF antibodies. Findings revealed that poly-His and SEKDEL labeling at the C-terminal is the most suitable for the optimized production of recombinant VEGF in N. benthamiana. Overall, VEGF protein has been successfully produced in plants that could be further characterized for its biological activity