Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant
文献类型：期刊论文
| 作者 | Zhang, Chun1,2; Liu, Yongdong2; Zhao, Dawei2; Li, Xiunan2; Yu, Rong1; Su, Zhiguo2 |
| 刊名 | PROTEIN EXPRESSION AND PURIFICATION
 |
| 出版日期 | 2014-03-01 |
| 卷号 | 95期号:1页码:195-203 |
| 关键词 | Tag-free rhTNF-alpha Purification E. coli expression Supernatant Two-step ion exchange chromatography |
| ISSN号 | 1046-5928 |
| 其他题名 | Protein Expr. Purif. |
| 中文摘要 | 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-alpha has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-alpha 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-alpha 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 beta-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-alpha was biologically active with a specific activity of approximately 2.0 x 10(7) U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-alpha from supernatant. (C) 2014 Elsevier Inc. All rights reserved. |
| 英文摘要 | 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-alpha has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-alpha 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-alpha 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 beta-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-alpha was biologically active with a specific activity of approximately 2.0 x 10(7) U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-alpha from supernatant. (C) 2014 Elsevier Inc. All rights reserved. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 研究领域[WOS] | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 关键词[WOS] | PICHIA-PASTORIS ; FUSION-PROTEIN ; AFFINITY TAGS ; BINDING ; SITE ; METALLOPROTEINASE ; ERYTHROPOIETIN ; CHROMATOGRAPHY ; DISINTEGRIN ; PEGYLATION |
| 收录类别 | SCI |
| 原文出处 | |
| 语种 | 英语 |
| WOS记录号 | WOS:000332192900028 |
| 公开日期 | 2014-05-06 |
| 版本 | 出版稿 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/8094]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 作者单位 | 1.Sichuan Univ, West China Sch Pharm, Minist Educ, Key Lab Drug Targeting & Drug Delivery Syst, Chengdu 610041, Peoples R China 2.Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China |
| 推荐引用方式 GB/T 7714 | Zhang, Chun,Liu, Yongdong,Zhao, Dawei,et al. Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant[J]. PROTEIN EXPRESSION AND PURIFICATION,2014,95(1):195-203. |
| APA | Zhang, Chun,Liu, Yongdong,Zhao, Dawei,Li, Xiunan,Yu, Rong,&Su, Zhiguo.(2014).Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant.PROTEIN EXPRESSION AND PURIFICATION,95(1),195-203. |
| MLA | Zhang, Chun,et al."Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant".PROTEIN EXPRESSION AND PURIFICATION 95.1(2014):195-203. |
入库方式： OAI收割
来源：过程工程研究所
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