ORIGINAL Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions Yulia Y. Stroylova • Tatiana Konnova • Yuriy F. Zuev • Jean-Marc Chobert • Yvan Choiset • Thomas Haertle ´ • Vladimir I. Muronetz Received: 11 December 2012 / Revised: 11 March 2013 / Accepted: 19 March 2013 / Published online: 6 April 2013 Ó Springer-Verlag Berlin Heidelberg 2013 Abstract In the present work, we proposed to create special sorbents for the study of protein–protein interac- tions, 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 cys- teines into the unfolded protein was not found to signifi- cantly 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 pro- posed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular gly- ceraldehyde-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 inter- acting 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 pro- tein-interacting partners, and the study of multi-protein complexes. Keywords Protein chromatography Á Thiol-Sepharose Á Protein–protein interactions Á Intrinsically unstructured proteins (IUP) Á Casein Abbreviations b-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 Introduction Over the last decade, methods of studying protein–protein interactions have changed significantly. Nowadays, the genetic engineering methods combined with the applica- tion of antibodies are mostly used instead of traditional approaches involving the whole spectrum of physico- chemical methods. For example, one of the developed methods of detecting partners in protein–protein interaction Y. Y. Stroylova Á V. I. Muronetz (&) Belozersky Institute of Physico-Chemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow State University, 119992 Moscow, Russia e-mail: vimuronets@belozersky.msu.ru Y. Y. Stroylova Á T. Konnova Á J.-M. Chobert Á Y. Choiset Á T. Haertle ´ Á V. I. Muronetz UR 1268 Biopolyme `res Interactions Assemblages, INRA, P. B. 71627, 44316 Nantes Cedex 3, France T. Konnova Á Y. F. Zuev Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P. B. 30, 420111 Kazan, Russian Federation 123 Chromatographia (2013) 76:621–628 DOI 10.1007/s10337-013-2463-1