کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359906 | 1542324 | 2016 | 38 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase
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کلمات کلیدی
FITCG-3-PDDFPTMs2DGERBCsPBSTGAPDHPVDFPTMRed blood cells - سلولهای قرمز خونPost translational modification - اصلاح پست ترجمه2-Dimensional gel electrophoresis - الکتروفورز ژل دو بعدیfluorescein isothiocyanate - فلوئورسین ایسوتیوسیاناتSubcellular localization - محلی سازی سلولPolyvinylidene fluoride - پلی وینیلیدین فلورایدglyceraldehyde-3-phosphate dehydrogenase - گلیسرالیدید-3-فسفات دهیدروژنازPlasmodium spp. - گونه های پلاسمودیوم
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase](/preview/png/8359906.png)
چکیده انگلیسی
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. â¼30-40 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 â¼Â 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 â¼27, â¼37 and â¼51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the â¼51 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 117, January 2016, Pages 17-25
Journal: Protein Expression and Purification - Volume 117, January 2016, Pages 17-25
نویسندگان
Prakash B. Sangolgi, Chinthapalli Balaji, Sneha Dutta, Nitin Jindal, Gotam K. Jarori,