Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders’ venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl-RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl-PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl-PLD1 was predicted as a “TIM beta/alpha-barrel”. The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were determined as 3.1 µg/mouse and 0.7 cm2 at 1 µg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.

The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli -expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.