Expression of novel lhmlt fusion protein using plant viral vector and study of its anticancer effect

Abstract

Melittin peptide is the main component of honey bee venom with the cytotoxic and anti-cancer effect which can affect healthy and cancerous cells simultaneously, so needed a proper solution for targeted use. The unique and overexpressed surface-receptor of the cancerous cells like Gonadotropin-Releasing Hormone Receptor (GnRHR) provide potential to deliver cytotoxic substances directly to cancerous cells. In this study, we attempted to produce a fusion protein (lhmlt) based on melittin and GnRH using plant system and investigate its effects on MCF7 (Breast), SH-SY5Y (Nerve) cancerous, and non-cancerous BMSCs cells. After assembling the lhmlt fusion model and structure prediction, to adapt to the parental subunit, the transient expression system based on the PVX viral vector and p19 suppressor was used to produce recombinant lhmlt in Nicotiana benthamiana plant. The lhmlt expression was confirmed by western blotting analysis and purified using cobalt chromatography column. The expression level was estimated to be 984 mg/kg plant fresh weight. Cell test results showed that the lhmlt fusion protein in low concentration inhibited the growth of cancer cells (MCF7:0.31 µg/ml, SH-SY5Y:0.15 µg/ml) with the GnRH receptor but the results were quite the opposite in normal cell without GnRHR so that the lhmlt in higher concentration (> 20 µg/ml) inhibited the BMSCs stem cells. In summary, the results indicate the targeted effect of lhmlt fusion protein on cancer cells and less damage to healthy cells, indicating the GnRH function on fusion protein structure that could herald the entry into the next stages of research. Transiently expression of lhmlt fusion protein based on melittin and GnRH in N.benthamiana and investigate its effect on cancer cells with GnRH-Receptor and normal cell without GnRH-Receptor.