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Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant
Alternative TitleProtein Expr. Purif.
Zhang, Chun1,2; Liu, Yongdong2; Zhao, Dawei2; Li, Xiunan2; Yu, Rong1; Su, Zhiguo2
2014-03-01
Source PublicationPROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
Volume95Issue:1Pages:195-203
AbstractFusing 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.
KeywordTag-free Rhtnf-alpha Purification E. Coli E.prE.sion Supernatant Two-step Ion Exchange Chromatography
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.pep.2013.12.012
URL查看原文
Indexed BySCI
Language英语
WOS KeywordPICHIA-PASTORIS ; FUSION-PROTEIN ; AFFINITY TAGS ; BINDING ; SITE ; METALLOPROTEINASE ; ERYTHROPOIETIN ; CHROMATOGRAPHY ; DISINTEGRIN ; PEGYLATION
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS IDWOS:000332192900028
Citation statistics
Cited Times:9[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/8094
Collection研究所(批量导入)
Affiliation1.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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