title: Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form.
abstract: Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100mM Tris buffer containing 6M n-propanol and 2M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6M n-propanol-based buffer containing 2M urea. Existence of native-like secondary structure of r-hGH in 6M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6M n-propanol and 2M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Surinder M
authors.initials: SM
authors.lastname: Singh
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Aparna
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Upadhyay
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Anupam
authors.initials: A
authors.lastname: Singh
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Lalit C
authors.initials: LC
authors.lastname: Garg
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India. Electronic address: amulya@nii.res.in.
authors.forename: Amulya K
authors.initials: AK
authors.lastname: Panda
mesh_terms: D000433:1-Propanol / Q000737:chemistry*; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D058849:Protein Refolding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone; D000433:1-Propanol