Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties.

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp.

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.