title: Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant.
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-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 2.0×10(7)U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-α from supernatant.
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: ydliu@home.ipe.ac.cn.
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Xiunan
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. Electronic address: yurong@scu.edu.cn.
authors.forename: Rong
authors.initials: R
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D051379:Mice; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha
keywords: E. coli expression; Purification; Supernatant; Tag-free rhTNF-α; Two-step ion exchange chromatography