Abstract
It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1α, interferon-γ, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturaing solubilization of some of these proteins we have tested.
Keywords: Non-denaturing solubilization, inclusion bodies, arginine, GFP, FGF-20
Current Pharmaceutical Biotechnology
Title: Non-Denaturing Solubilization of Inclusion Bodies
Volume: 11 Issue: 3
Author(s): Kouhei Tsumoto, Ryota Abe, Daisuke Ejima and Tsutomu Arakawa
Affiliation:
Keywords: Non-denaturing solubilization, inclusion bodies, arginine, GFP, FGF-20
Abstract: It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1α, interferon-γ, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturaing solubilization of some of these proteins we have tested.
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Cite this article as:
Tsumoto Kouhei, Abe Ryota, Ejima Daisuke and Arakawa Tsutomu, Non-Denaturing Solubilization of Inclusion Bodies, Current Pharmaceutical Biotechnology 2010; 11 (3) . https://dx.doi.org/10.2174/138920110791111924
DOI https://dx.doi.org/10.2174/138920110791111924 |
Print ISSN 1389-2010 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4316 |
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