Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity

Christine Joy I Bulaon; Balamurugan Shanmugaraj; Yamin Oo; Kaewta Rattanapisit; Taksina Chuanasa; Chatchai Chaotham; Waranyoo Phoolcharoen

doi:10.1016/j.btre.2020.e00514

Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity

Biotechnol Rep (Amst). 2020 Aug 15:27:e00514. doi: 10.1016/j.btre.2020.e00514. eCollection 2020 Sep.

Authors

Christine Joy I Bulaon¹, Balamurugan Shanmugaraj^{1

2}, Yamin Oo³, Kaewta Rattanapisit^{1

2}, Taksina Chuanasa¹, Chatchai Chaotham^{3

4}, Waranyoo Phoolcharoen^{1

2}

Affiliations

¹ Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
² Research Unit for Plant-produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.
³ Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 10330, Bangkok, Thailand.
⁴ Cell-based Drug and Health Products Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 10330, Bangkok, Thailand.

Abstract

Human vascular endothelial growth factor (VEGF) is a potent pro-angiogenic growth factor essential for wound healing. Due to its potential applications, many expression strategies have been developed to produce high levels of VEGF. Here, we have optimized the expression conditions for the production of recombinant VEGF in Nicotiana benthamiana by using a geminiviral vector. Four different expression constructs that differ by the location of a C- or N-terminal histidine tag and SEKDEL sequence were developed and utilized for plant transient expression. The recombinant VEGF was further purified by using affinity chromatography and confirmed by SDS-PAGE and Western blotting probed with anti-VEGF antibody. Furthermore, our results showed that the recombinant VEGF in all tested concentrations did not exhibit any cytotoxic effect on HaCaT cells and induced cell migration in vitro. These findings show that the plant-produced VEGF has the potential to be used in regenerative medicine and cosmetic industry.

Keywords: Human vascular endothelial growth factor; Molecular farming; Nicotiana benthamiana; Transient expression.