Manabayeva SA

National Center for Biotechnology of Republic of Kazakhstan, Kazakhstan

Title: Tomato bushy stunt virus-based vector for transient expression of human G-CSF in Nicotiana benthamiana plants



The use of plant viral vectors for transient expression of foreign proteins in plants offers a useful tool for the production of proteins such as biopharmaceuticals. Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage in a dose-dependent manner. The objective of this work was the development of an advanced Tomato bushy stunt virus (TBSV)based protein production system to produce human G-CSF in non-transgenic plants. The fact that the TBSV genome encodes the p19 protein, capable of inhibition of posttranscriptional silencing and enhancing expression levels for each gene in the viral RNA (including heterologous ones), is a significant advantage of the TBSV vector system. We evaluated the potential of a TBSV vector system for efficient expression of the recombinant human G-CSF protein in Nicotiana benthamiana plants. For this purpose we developed a TBSV derived viral vector driven by the CaMV 35S promoter that can be delivered as DNA. In this vector the CP gene was replaced by the codon optimized G-CSF (174 a.a., molecular weight 18,6 kDa) gene that was synthesized by a PCR-based gene synthesis method. To facilitate purification of the rhG-CSF from plant materials the protein sequence was modified by addition of the short amino acid tag (6 x His). The viral vector was delivered into 4-5 week old N.benthamiana plants by agroinfiltration. Four days after infiltration expression of rhC-CSF was verified by protein extraction followed by western blots procedures. The rhG-CSFx6His was isolated using metal affinity chromatography on Ni2NTA agarose. The concentration of purified rhG-CSF was determined by ELISA. The preliminary results indicate that the TBSV - based protein production system is suitable for transient production of biopharmaceuticals in plants.