Selective cross-linking of interacting proteins using self-labeling tags.
We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We validated our approach by confirming a number of interactions through selective cross-linking and showed that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein complexes and the differentiation between strong and weak protein-protein interactions.
Gautier
Arnaud
A
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
Nakata
Eiji
E
Lukinavicius
Grazvydas
G
Tan
Kui-Thong
KT
Johnsson
Kai
K
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013194:Staining and Labeling
D003432:Cross-Linking Reagents; D011506:Proteins; D012967:Sodium Dodecyl Sulfate