Apoplastic Production of Recombinant AntiVEGF Protein Using Plant-Virus Transient Expression Vector.

Targeting of vascular endothelial growth factor (VEGF) using AntiVEGF can be a promising approach for angiogenesis inhibition and cancer therapy. In this study, we direct AntiVEGF recombinant protein accumulation to cucurbit plant apoplast using a suitable signal (Pr1b) sequence. After assembling the target gene construct and cloning into the expression vector, we infected the plants with the resulting pZYMV-AntiVEGF viral vector. Transcription of the target gene was confirmed with RT-PCR assays. The apoplast-targeted AntiVEGF recombinant protein was detected in infected plants by Dot-blot, western blot, and ELISA analysis. AntiVEGF protein accumulation in the apoplast resulted in levels of 1.2% of TSP (Total Soluble Protein) that demonstrated a two-order increase compared to the cytoplasm-targeted protein. After purification of AntiVEGF protein using aqueous two-phase system (ATPS), purified protein was analyzed with MTT assay. Our results reveal that production of biologically active and correctly processed apoplast-targeted AntiVEGF recombinant protein is possible in plant apoplast. The low level of cytoplasm-targeted AntiVEGF recombinant protein might result from the degradation of improperly folded protein.

Identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect Sphodromantis viridis.

Antimicrobial peptides are members of the immune system that protect the host from infection. In this study, a potent and structurally novel antimicrobial peptide was isolated and characterized from praying mantis Sphodromantis viridis. This 14-amino acid peptide was purified by RP-HPLC. Tandem mass spectrometry was used for sequencing this peptide, and the results showed that the peptide belongs to the Mastoparan family. The peptide was named Mastoparan-S. Mastoparan-S demonstrated that it has antimicrobial activities against a broad spectrum of microorganisms (Gram-positive and Gram-negative bacteria and fungi), and it was found to be more potent than common antibiotics such as kanamycin. Mastoparan-S showed higher antimicrobial activity against Gram-negative bacteria compared to Gram-positive ones and fungi. The minimum inhibitory concentration (MIC) values of Mastoparan-S are 15.1-28.3 µg/ml for bacterial and 19.3-24.6 µg/ml for fungal pathogens. In addition, this newly described peptide showed low hemolytic activity against human red blood cells. The in vitro cytotoxicity of Mastoparan-S was also evaluated on monolayer of normal human cells (HeLa) by MTT assay, and the results illustrated that Mastoparan-S had significant cytotoxicity at concentrations higher than 40 µg/ml and had no any cytotoxicity at the MIC (≤30 µg/ml). The findings of the present study reveal that this newly described peptide can be introduced as an appropriate candidate for treatment of topical infection.