کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843395 | 1069232 | 2009 | 5 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris
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موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris](/preview/png/10843395.png)
چکیده انگلیسی
Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5 mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS-PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen α-chain. The enzyme also degraded fibrinogen β-chain to a lesser extent, while showing no degradation toward the γ-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombin-like enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate Nα-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 °C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 66, Issue 2, August 2009, Pages 138-142
Journal: Protein Expression and Purification - Volume 66, Issue 2, August 2009, Pages 138-142
نویسندگان
Daping Yang, Mingli Peng, Hua Yang, Qing Yang, Jianqiang Xu,