Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent

Abstract

BackgroundFormation 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.ResultsHuman 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.ConclusionsTrifluoroethanol 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0504-9) contains supplementary material, which is available to authorized users.

Solubilization of Human Interferon β-1b Inclusion Body Proteins by Organic Solvents

“…Given that prokaryotic cells are not able to form disulfide bonds and also cannot perform post-translational processes, the expressed recombinant protein does not have its own natural structure as a result and forms intracellular insoluble particles known as "Inclusion Bodies" (IB) which contain native-like secondary structure, condensed, classically inactive and insoluble proteins. [7][8][9] In other words, IBs are the result of the recombinant protein overexpression in the expression system. 10,11 Production of bio-drugs in E. coli as a host cell has many challenges due to the formation of IBs.…”

Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

“…Despite the extensive experience and widespread use of recombinant technology, many challenges remain when generating recombinant variants of native proteins. Escherichia coli has been the host of choice for manufacturing of numerous recombinant proteins, and overexpression of the target proteins in E. coli leads to the accumulation of partially folded protein aggregates, called inclusion bodies (IBs), which contain native‐like secondary structures and classically lack biological activity . Subsequent to bacterial cell rupture or disruption, IBs isolation from the whole cell lysate and reduction of impurities by washing with buffers containing low concentrations of detergents, IB proteins must be solubilized and refolded into an active conformation .…”

“…Proteins expressed as inclusion bodies are typically solubilized in high concentrations of chaotropic agents, such as urea and guanidinium chloride and this process results in denaturation of the native‐like secondary structures in the proteins. Complete unfolding of protein molecules in IBs throughout solubilization enhances the opportunity of aggregation during refolding and consequently leads to low recovery of bioactive protein . For instance, the large‐scale strategy for production of the recombinant plasminogen activator, streptokinase, via expression as IBs in E. coli has been depicted in Fig.…”

“…Escherichia coli has been the host of choice for manufacturing of numerous recombinant proteins, and overexpression of the target proteins in E. coli leads to the accumulation of partially folded protein aggregates, called inclusion bodies (IBs), which contain native-like secondary structures and classically lack biological activity. [1][2][3] Subsequent to bacterial cell rupture or disruption, IBs isolation from the whole cell lysate and reduction of impurities by washing with buffers containing low concentrations of detergents, IB proteins must be solubilized and refolded into an active conformation. [4][5][6][7] However, solubilization of inclusion bodies and refolding of the solubilized proteins into bioactive form is a challenging process since the contained protein molecules are in an aggregate state.…”

“…Complete unfolding of protein molecules in IBs throughout solubilization enhances the opportunity of aggregation during refolding and consequently leads to low recovery of bioactive protein. 3,9,10 For instance, the large-scale strategy for production of the recombinant plasminogen activator, streptokinase, via expression as IBs in E. coli has been depicted in Fig. 1.…”