A novel recombinant javanicin with dual antifungal and anti-proliferative activities

Abstract

Resistance to common drugs by microorganisms and cancers has become a major issue in modern healthcare, increasing the number of deaths worldwide. Novel therapeutic agents with a higher efficiency and less side effects for the treatment of certain diseases are urgently needed. Plant defensins have an integral role in a hosts’ immune system and are attractive candidates for combatting drug-resistant microorganisms. Interestingly, some of these defensins also showed great potential due to their cytotoxic activity toward cancer cells. In this study, a defensin encoding gene was isolated from five legume seeds using 3′ rapid amplification of cDNA ends (3′ RACE) with degenerate primers and cDNA cloning strategies. Bioinformatic tools were used for in silico identification and the characterization of new sequences. To study the functional characteristics of these unique defensins, the gene encoded for Sesbania javanica defensin, designated as javanicin, was cloned into pTXB-1 plasmid and expressed in the Escherichia coli Origami 2 (DE3) strain. Under optimized conditions, a 34-kDa javanicin-intein fusion protein was expressed and approximately 2.5–3.5 mg/L of soluble recombinant javanicin was successfully extracted with over 90% purity. Recombinant javanicin displayed antifungal properties against human pathogenic fungi, including resistant strains, as well as cytotoxic activities toward the human breast cancer cell lines, MCF-7 & MDA-MB-231. Recombinant javanicin holds great promise as a novel therapeutic agent for further medical applications.

Fungicidal Activity of Recombinant Javanicin against Cryptococcus neoformans Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes

“…So that the measurement of capsule thickness or determination of antifungal activity in capsular and acapsular strains in the presence of r-javanicin might be further performed for supporting the effect of peptide. In addition, our previous study revealed that r-javanicin induced the death of breast cancer cells (MCF-7) via apoptotic pathway [15]. Although it is still unclear about the correlation between the antifungal and anticancer activity of this novel peptide, it is possible that r-javanicin might directly target mitochondria where the induction of apoptosis is originated.…”

“…Along the way, the study of the mechanism of actions of these candidate peptides has become one of the indispensable processes for further development for therapeutic potential. Javanicin, a class of defensin derived from the seed of S. javanica, was previously identified and heterologous expressed in E. coli [15]. The peptide exhibited potent anti-proliferative activity against breast cancer cells and antifungal activities in both human pathogenic yeast and mold, including fluconazole sensitive and fluconazole-resistant Candida albicans, C. neoformans, and Trichophyton rubrum [15].…”

“…Javanicin, a class of defensin derived from the seed of S. javanica, was previously identified and heterologous expressed in E. coli [15]. The peptide exhibited potent anti-proliferative activity against breast cancer cells and antifungal activities in both human pathogenic yeast and mold, including fluconazole sensitive and fluconazole-resistant Candida albicans, C. neoformans, and Trichophyton rubrum [15]. However, the mode of action by which javanicin exerts its antifungal effect is still unknown.…”

“…The peptide comprises of 47 amino acids with four disulfide bonds and is predicted to adopt a single alpha-helix and triplestranded antiparallel beta-sheet structure. From our previous study, recombinant javanicin (r-javanicin) was produced using E. coli expression system [15]. The purified peptide was examined and exhibited potent antifungal activity against human fungal pathogens including C. neoformans.…”

Immunomodulatory Proteins in Tick Saliva From a Structural Perspective

“…While that could be achieved using eukaryotic expression systems, tick proteins are often highly glycosylated ( Tyson et al., 2007 ; Dias et al., 2009 ), which could hinder crystallization for X-ray crystallography and lead to spectral line broadening in NMR spectroscopy experiments. The correct disulfide connectivity could be achieved in E.coli using Origami strains, which greatly enhances disulfide bond formation due to mutated thioredoxin and glutathione reductases ( Orrapin et al., 2019 ). In general, expressed proteins must be refolded in the presence of a redox couple, but often it requires tedious screening for optimal conditions and should be performed under the tight control of LC-MS analysis to obtain the homogeneous protein in the correct conformation.…”