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Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with K m and V max values of 3.5 mM and 10.4 μmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 μg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 μg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy. [Display omitted] • A novel homodimeric L-amino acid oxidase with two subunits of 70 kDa was purified from Egyptian Cobra (Naja haje) venom. • The purified L-amino acid oxidase is optimally active at pH 8 and 37 °C with L-Leucine as a substrate. • Naja haje - L-amino acid oxidase displays anti-bacterial effect against several bacterial strains. • The purified L-amino acid oxidase induces apoptotic activity against hepatic cancer cell lines. • This study offers insight into L-amino acid oxidase purified from Naja haje venom as a promising cancer drug candidate. [ABSTRACT FROM AUTHOR]

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His 6 -tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs. [Display omitted] • α-cobratoxin produced in bacteria or yeast is not completely identical to the native toxin structure • α-cobratoxin produced in yeast has a similar circular dichroism spectrum as the native toxin and binds the target receptor • Solubilization tags aid in enhancing the expression of soluble protein, but removal negatively affects net yield • E. coli and K. phaffii -expressed α-cobratoxin retains functional binding to its target receptor. • Recombinant toxins can be used to advance antivenom research and the development of toxin-derived therapeutics. [ABSTRACT FROM AUTHOR]