Abstract
In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.
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Abbreviations
- β-CN:
-
Beta-casein
- bME:
-
Beta-mercaptoethanol
- DLS:
-
Dynamic light scattering
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediaminetetraacetic acid
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- IUP:
-
Intrinsically unstructured protein
- MQ:
-
MilliQ water
- PBS:
-
Phosphate-buffered saline
- PrP:
-
Prion protein
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Acknowledgments
The authors acknowledge the support from French Embassy in of form of a Ph.D in co-tutelle funding for T.K. and Y.Y.S. and the funding of “Chaire régionale de chercheur étranger” by Région Pays de Loire for V.I.M. The authors acknowledge the support of the Russian Foundation for Basic Researches (12-08-31212, 12-08-33063, 11-04-01350-a, 09-04-92740-NNIOM_a), and the Russian Federation President Grant for young scientists to Y.Y.S. (MК-877.2012.4). This work was partly supported by the Russian Academy of Sciences under the Program ‘‘Molecular and Cellular Biology” and by French Embassy to Y.F.Z.
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Stroylova, Y.Y., Konnova, T., Zuev, Y.F. et al. Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions. Chromatographia 76, 621–628 (2013). https://doi.org/10.1007/s10337-013-2463-1
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DOI: https://doi.org/10.1007/s10337-013-2463-1