کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843492 | 1069262 | 2005 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30Â mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34Â kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 42, Issue 2, August 2005, Pages 246-254
Journal: Protein Expression and Purification - Volume 42, Issue 2, August 2005, Pages 246-254
نویسندگان
Yan-Li Zhang, Song-Sen Chen, Ke-Gong Yang, Lin Su, Yan-Chun Deng, Chang-Zheng Liu,