Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography

Autor: Victoria Veroli, Mónica Marín, Jenny Saldaña, Elisa Melian, Laura Domínguez, Mario Señorale, Ramiro Teixeira, Beatriz Munguía, Mahia Minteguiaga
Rok vydání: 2017
Předmět:
0301 basic medicine
Male
Spectrometry
Mass
Electrospray Ionization
Saccharomyces cerevisiae Proteins
Swine
Recombinant Fusion Proteins
030231 tropical medicine
Immunology
Saccharomyces cerevisiae
Sheep Diseases
macromolecular substances
Biology
Chromatography
Affinity
law.invention
03 medical and health sciences
Mice
0302 clinical medicine
Affinity chromatography
Mesocestoides
Microtubule
law
Tubulin
Escherichia coli
Animals
Amino Acid Sequence
Glutathione Transferase
chemistry.chemical_classification
Sheep
Glutamate dehydrogenase
General Medicine
biology.organism_classification
Cestode Infections
Molecular biology
030104 developmental biology
Infectious Diseases
Enzyme
Biochemistry
chemistry
Spectrometry
Mass
Matrix-Assisted Laser Desorption-Ionization
Recombinant DNA
biology.protein
Parasitology
Haemonchus
Haemonchiasis
Microtubule-Associated Proteins
Haemonchus contortus
Zdroj: Experimental parasitology. 182
ISSN: 1090-2449
Popis: Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 μg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites.
Databáze: OpenAIRE
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