کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360696 | 1542346 | 2014 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant
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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: Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant](/preview/png/8360696.png)
چکیده انگلیسی
Fusing affinity tag at N-terminus was reported to decrease the biological activity of the recombinant human tumor necrosis factor alpha. Although preparation of tag-free rhTNF-α has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-α was almost equally synthesized by Escherichia coli in both soluble and insoluble forms. A two-step ion exchange chromatography, DEAE-Sepharose combined with CM-Sepharose, was developed to purify the soluble specie from supernatant after cell lysis. Native PAGE and HP-SEC showed the rhTNF-α extracted from supernatant existed in a homogeneous form. HP-SAX and SDS-PAGE analysis demonstrated the purity of the final fraction was over 98% with a very high recovery of 75%. Circular dichroism spectrum demonstrated that β-sheet structure was dominant and fluorescence analysis suggested no dramatic exposure of aromatic amino acid residues on the protein surface. Bioassay indicated that purified rhTNF-α was biologically active with a specific activity of approximately 2.0 Ã 107 U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-α from supernatant.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 95, March 2014, Pages 195-203
Journal: Protein Expression and Purification - Volume 95, March 2014, Pages 195-203
نویسندگان
Chun Zhang, Yongdong Liu, Dawei Zhao, Xiunan Li, Rong Yu, Zhiguo Su,