Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.
BACKGROUND
Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form.


RESULTS
Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize inclusion bodies of a number of proteins.


CONCLUSIONS
Trifluoroethanol was found to be a suitable mild solubilization agent for bacterial inclusion bodies. Fully functional, bioactive human growth hormone was recovered in high yield from inclusion bodies using trifluoroethanol based solubilization buffer. It was also observed that trifluoroethanol has potential to solubilize inclusion bodies of different proteins.
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Vaibhav
V
Upadhyay
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Anupam
A
Singh
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Divya
D
Jha
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Akansha
A
Singh
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India. amulya@nii.res.in.
Amulya K
AK
Panda
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014270:Trifluoroethanol / Q000737:chemistry*
D011994:Recombinant Proteins; D019382:Human Growth Hormone; D014270:Trifluoroethanol
Human growth hormone; Inclusion bodies; Mild solubilization; Refolding; Trifluoroethanol