Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase.
Protein Expr Purif
; 117: 17-25, 2016 Jan.
Article
em En
| MEDLINE
| ID: mdl-26341815
ABSTRACT
Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. â¼ 30-40 mg pure recombinant enzyme with a specific activity of 12.6 units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH â¼ 9. Kinetic measurements gave KmNAD=0.28 ± 0.3 mM and KmG3P=0.25 ± 0.03 mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW â¼ 27, â¼ 37 and â¼ 51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the â¼ 51 kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membranes) examined had Gapdh associated with them. Each fraction had multiple molecular species associated with them. Such species could arise only by multiple post-translational modifications. Structural heterogeneity observed among molecular species of PyGapdh and their diverse subcellular distribution, supports the view that Gapdh is likely to have multiple non-glycolytic functions in the parasite and could be an effective target for anti-malarial chemotherapeutics.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plasmodium falciparum
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Plasmodium yoelii
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Proteínas de Protozoários
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Gliceraldeído-3-Fosfato Desidrogenases
Limite:
Animals
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Humans
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Índia