title: One-step refolding and purification of recombinant human tumor necrosis factor-α (rhTNF-α) using ion-exchange chromatography.
abstract: Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor-α (rhTNF-α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF-α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF-α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10(8) IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF-α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF-α refolding in terms of specific bioactivity and mass recovery.
authors.affiliation: Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi'an, 710069, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Wenxuan
authors.initials: W
authors.lastname: Ren
authors.forename: Dong
authors.initials: D
authors.lastname: Gao
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.forename: Quan
authors.initials: Q
authors.lastname: Bai
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha
keywords: inclusion bodies; ion exchange chromatography; protein folding liquid chromatography; protein refolding; recombinant human tumor necrosis factor-α