Knowledge Management System Of Institute of process engineering,CAS
| Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant | |
| Alternative Title | Protein Expr. Purif. |
| Zhang, Chun1,2; Liu, Yongdong2; Zhao, Dawei2; Li, Xiunan2; Yu, Rong1; Su, Zhiguo2 | |
| 2014-03-01 | |
| Source Publication | PROTEIN EXPRESSION AND PURIFICATION
 |
| ISSN | 1046-5928 |
| Volume | 95Issue:1Pages:195-203 |
| Abstract | 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.; 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. |
| Keyword | Tag-free Rhtnf-alpha Purification E. Coli E.prE.sion Supernatant Two-step Ion Exchange Chromatography |
| Subtype | Article |
| WOS Headings | Science & Technology ; Life Sciences & Biomedicine |
| DOI | 10.1016/j.pep.2013.12.012 |
| URL | 查看原文 |
| Indexed By | SCI |
| Language | 英语 |
| WOS Keyword | PICHIA-PASTORIS ; FUSION-PROTEIN ; AFFINITY TAGS ; BINDING ; SITE ; METALLOPROTEINASE ; ERYTHROPOIETIN ; CHROMATOGRAPHY ; DISINTEGRIN ; PEGYLATION |
| WOS Research Area | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS Subject | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS ID | WOS:000332192900028 |
| Citation statistics | |
| Document Type | 期刊论文 |
| Version | 出版稿 |
| Identifier | http://ir.ipe.ac.cn/handle/122111/8094 |
| Collection | 研究所（批量导入） |
| Affiliation | 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 |
| Recommended Citation 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. |
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| Facile purification （1243KB） | 限制开放 | CC BY-NC-SA | Application Full Text | |||
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