کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5516122 1542304 2017 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Isolation of recombinant human untagged glyceraldehyde-3-phosphate dehydrogenase from E. coli producer strain
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Isolation of recombinant human untagged glyceraldehyde-3-phosphate dehydrogenase from E. coli producer strain
چکیده انگلیسی


- Untagged human glyceraldehyde-3-phosphate dehydrogenase was expressed in E. coli cells.
- The enzyme was isolated using sulfate ammonium fractionation and gel chromatography.
- 600 ml of the culture yields 2 mg of recombinant protein with high enzymatic activity.

The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass). The specific activity of the freshly purified hGAPDH constitutes 117 ± 5 μmol NADH/min per mg protein (pH 9.0, 22 °C), which is close to the specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase determined under the same conditions and several times exceeds the specific activity of his-tagged GAPDH preparations. The high enzymatic activity suggests that the recombinant enzyme retains its native structure. The described procedure may be useful for researchers who need a preparation of native hGAPDH without admixture of misfolded forms for their investigations.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 137, September 2017, Pages 1-6
نویسندگان
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