Abstract
A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×107 IU/mg.
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References
Lanckriet, H. and A. P. J. Meddelberg (2004) Continuous chromatographic protein refolding.J. Chromatogr. A. 1022: 103–113.
De Bernardez Clark, E. (2001) Protein refolding for industrial processes.Curr. opin. Biotechnol. 12: 202–207.
Lilie, H., E. Schwarz and R. Rudolph (1998) Advances in refolding of proteins produced inE. coli.Curr. Opin. Biotechnol. 9: 497–501.
Rudolph, R. and H. Lilie (1996)In vitro folding of inclusion body proteins.FASEB J. 10: 49–56.
Cui, Z. F., Y. X. Guan, and S. J. Yao (2004) A temperature-sensitive hydrogel refolding system: Preparation of poly(N-isopropyl acrylamide) and its application in lysozyme refolding.Chinese J. Chem. Eng. 12: 556–560.
Rudlph, R., G. Bohm, H. Lilie and R. Jaenicke (1997)In Protein Function: A Practical Approach. 2nd ed., pp. 57–99. IRL Press, Oxford, UK.
Park, S. J., K. Ryu, C. W. Suh, Y. G. Chai, O. B. Kwon, S. K. Park, and E. K. Lee (2002) Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin.Biotechnol. Bioprocess Eng. 7: 1–5.
Creighton, T. E. (1990)US Patent 4,977,248.
Fahey, E. M., J. B. Chaudhuri and P. Binding (2000) Refolding and purification of a urokinase plasminogen activator fragment by chromatography.J. Chromatogr. B. 737: 225–235.
Rogl, H., K. Kosemund, W. Kuhlbrandt and I. Collinson (1998) Relolding ofEscherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography.FEBS Letters 432: 21–26.
Geng, X. D. and X. Q. Chang (1992) High-performance hydrophobic interaction chromatography as a tool for protein refolding.J. Chromatogr. A. 599: 185–194.
Batas, B. and J. B. Chaudhuri (1999) Considerations of sample application and elution during size-exclusion chromatography-based protein refolding.J. Chromatogr. A. 864: 229–236.
Pestka, S., A. J. Langers, C. K. Zoon and C. E. Samuel (1987) Interferons and their actions.Annual Review Biochemistry 56: 727–777.
Zhang, D. Z., S. H. Wu, Y. D. Hou and C. Z. Su (1989) The purification of recombinant human interferon-γ.Chinese J. Virology 5: 37–40.
Bradford, M. M. (1976) A rapid and sensitive method for the quantization of protein utilizing the principles of protein-dye binding.Anal. Biochem. 72: 248–254.
Du, P. (1984)Medical Interferon. pp. 231–307. Liberation Army Publishing House, Beijing, China.
Laemmli, U. K. (1970) Detection of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680–685.
Gu, Z. Y., Z. G. Su and J. C. Janson (2001) Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding.J. Chromatogr. A. 918: 311–318.
Li, M., G. F. Zhang and Z. G. Su (2002) Dual gradient ion-exchange chromatography improved refolding yield of lysozyme.J. Chromatogr. A. 959: 113–120.
Kim, C. S. and E. K. Lee (2000) Effects of operating parameters inin vitro renaturation of a fusion protein of human growth hormone and glutathiones transferase for inclusion body.Process Biochemistry 36: 111–117.
Gao, Y. G., Y. X. Guan, S. J. Yao and M. G. Cho (2005) On-column refolding of recombinant human interferon-γ with an immobilized chaperone fragment.Biotechnol. Prog. 19: 915–920.
Gao, Y. G., Y. X. Guan, S. J. Yao and M. G. Cho (2002) Refolding of lysozyme at high concentration in batch and fed-batch operation.Korean J. Chem. Eng. 19: 871–875.
Gao, Y. G., Y. X. Guan, S. J. Yao and M. G. Cho (2003) Lysozyme refolding at high concentration by dilution and size-exclusion chromatography.J. Zhejiang University Science 4: 136–141.
Cho, T. H., S. J. Ahn and E. K. Lee (2002) Refolding of protein inclusion bodies directly fromE. coli homogenate using expanded bed adsorption chromatography.Bioseparation 10: 189–196.
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Guan, YX., Pan, HX., Gao, YG. et al. Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography. Biotechnol. Bioprocess Eng. 10, 122–127 (2005). https://doi.org/10.1007/BF02932581
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DOI: https://doi.org/10.1007/BF02932581