Abstract
A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC–MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.
Highlights
Antimicrobial compounds are of high clinical value for the treatment of bacterial infections and infections caused by several fungi as w ell[12]
Napin was precipitated by ammonium sulfate from the crude extract of Eruca sativa (Fig. 1A)
Napin protein remained in supernatant after 50% (w/v) (NH4)2SO4 saturation constant while subsequent 70% precipitated the protein in pellet
Summary
Antimicrobial compounds are of high clinical value for the treatment of bacterial infections and infections caused by several fungi as w ell[12]. Rocket Salad (Eruca sativa Miller), locally known as Taramira; is an annual herb and belongs to the family Brassicaceae (Cruciferae). Eruca seeds possess various proteins, glucosinolates, vitamins A and C, flavonoids, erucic acid and a relatively high oil c ontent[17,21]. It is commonly used as animal feed in Asia, in India and Pakistan. In view of its potential medicinal uses, it is hypothesized that Rocket Salad might have antimicrobial proteins/peptides in their seeds/leaves which can be exploited for the development of anti-cancerous drug after detailed understanding of its structure with subsequent characterization. Present study describes the structural insights and thorough functional characterization of a napin, which was identified and purified from seeds of E. sativa
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