title: Refolding of recombinant human interferon alpha-2a from Escherichia coli by urea gradient size exclusion chromatography.
abstract: Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon alpha-2a (rhLFNalpha-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhLFNalpha-2a would pass along the 8.0-3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 x 10(8) IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhLFNalpha-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhl FNalpha-2a refolding in terms of specific activity and mass recovery.
authors: College of Life Science, Qufu Normal University, Qufu 273165, China. gaofeiqf@yahoo.cn
authors: F
authors: F
authors: Gao
authors: L
authors: L
authors: Shi
authors: L X
authors: LX
authors: Xu
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014508:Urea / Q000737:chemistry*
chemical_list: D016898:Interferon-alpha; D011994:Recombinant Proteins; D014508:Urea; D005978:Glutathione