Journal Article

Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris

da Rocha, Surza Lucia Gonçalves, Almeida, Rodrigo Volcan, Vieira, Saulo Martins, Perales, Jonas, Neves-Ferreira, Ana Gisele da Costa

2017

Overview

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Affinity chromatography / Amino Acid Sequence / Animals / Ascomycota / Biochemistry / Biodegradation / Biological activity / Biological properties / Biology and Life Sciences / Bites (Injuries) / Blood Proteins - biosynthesis / Blood Proteins - chemistry / Bothrops / Brazil / Care and treatment / Cell culture / Cell Line / Components / Countries / Culture media / Cytotoxicity / Degradation / Fermentation / Funding / Gene expression / Genetic aspects / Glycoproteins / Glycosylation / Growth / Health aspects / Inhibitors / Ion exchange / Laboratories / Mass Spectrometry / Mass spectroscopy / Medicine and Health Sciences / Mice / Opossums / Phospholipase / Phospholipase A2 / Phospholipase A2 Inhibitors - chemistry / Phospholipases A2 - chemistry / Physical Sciences / Pichia / Pichia pastoris / Protease inhibitors / Protein folding / Proteinase inhibitors / Proteins / Proteolysis / Public health / Recombinant Proteins - biosynthesis / Recombinants / Reptilian Proteins - chemistry / Research and Analysis Methods / Roles / Science / Scientific imaging / Serine / Serine proteinase / Serum / Snake bites / Snake Venoms - chemistry / Supervision / Tissue / Toxicity / Toxins / Tropical climate / Tropical diseases / Venom / Western blotting / Yeast / Yeasts / Yields