Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris
| Autor: | Surza Lucia Gonçalves da Rocha, Saulo Martins Vieira, Rodrigo Volcan Almeida, Jonas Perales, Ana G.C. Neves-Ferreira |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
PNGase F Glycobiology Reptilian Proteins Toxicology Pathology and Laboratory Medicine Biochemistry Physical Chemistry Mass Spectrometry Pichia Mice Medicine and Health Sciences Toxins Bothrops Snakebite Gel Electrophoresis Staining biology lcsh:Public aspects of medicine Snakes Blood Proteins Recombinant Proteins Squamates Chemistry Molecular Mass Infectious Diseases Physical Sciences Vertebrates Brazil Snake Venoms Research Article Neglected Tropical Diseases Silver Staining lcsh:Arctic medicine. Tropical medicine lcsh:RC955-962 Phospholipase A2 Inhibitors Myotoxin Toxic Agents Heterologous Electrophoretic Staining Research and Analysis Methods Pichia Pastoris Cell Line Pichia pastoris Electrophoretic Techniques 03 medical and health sciences Affinity chromatography Animals Amino Acid Sequence Glycoproteins Serine protease Venoms Organisms Fungi Public Health Environmental and Occupational Health Biology and Life Sciences Proteins Reptiles lcsh:RA1-1270 Opossums Tropical Diseases biology.organism_classification Molecular biology Yeast Phospholipases A2 030104 developmental biology Chemical Properties Specimen Preparation and Treatment Amniotes biology.protein Heterologous expression |
| Zdroj: | PLoS Neglected Tropical Diseases PLoS Neglected Tropical Diseases, Vol 11, Iss 7, p e0005829 (2017) |
| ISSN: | 1935-2735 |
| DOI: | 10.1371/journal.pntd.0005829 |
| Popis: | 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. Author summary Snakebite envenomation causes medical emergencies that, depending on the species responsible for the bite, involve different organs and tissues. Envenomation by snakebite is a worldwide problem, and Brazil presents a high incidence of Bothrops bites. Bothrops venoms cause pathological alterations with prominent local effects, such as edema, blistering, hemorrhage, dermonecrosis and myonecrosis, usually followed by poor tissue regeneration and permanent sequelae. Bleeding, coagulopathy, cardiovascular shock and renal failure are typical systemic effects of these venoms. The clinical treatment for snakebite envenoming is intravenous administration of the specific antivenom. However, serotherapy does not efficiently protect against local tissue damage. Additional challenges faced by classical antivenom therapy include the wide antigenic variation of venoms across species and even within the same snake species and the frequent occurrence of adverse reactions that are associated with the administration of immunobiologicals. The development of new effective toxin inhibitors based on the structure of natural antiophidic proteins is an attractive therapeutic alternative. DM64 is a myotoxin inhibitor that was isolated from opossum serum, and its expression as a recombinant protein is paramount to the characterization of its structure-function relationship, an essential step toward the development of alternative strategies to better manage bothropic snakebite envenomations. |
| Databáze: | OpenAIRE |
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