Selective cross-linking of interacting proteins using self-labeling tags

Journal of the American Chemical Society
Arnaud GautierKai Johnsson

Abstract

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.

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Citations

Sep 2, 2011·Accounts of Chemical Research·Chaoran Jing, Virginia W Cornish
Sep 26, 2013·Nanoscale·Katrine R RostgaardKaren L Martinez
May 16, 2012·FEBS Letters·Mateusz Putyrski, Carsten Schultz
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Oct 30, 2010·Current Opinion in Biotechnology·Marlon J Hinner, Kai Johnsson
Oct 30, 2010·Current Opinion in Chemical Biology·David Rabuka
Apr 23, 2013·Chemistry & Biology·Dominik ErhartMatthias P Wymann
Jan 22, 2013·Current Biology : CB·Gražvydas LukinavičiusKai Johnsson
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Jul 22, 2014·European Journal of Medicinal Chemistry·Xiaoli SunMing-Qun Xu
Jun 13, 2012·Angewandte Chemie·Anna Rutkowska, Carsten Schultz
Sep 12, 2012·Chembiochem : a European Journal of Chemical Biology·Matthias P Wymann, Carsten Schultz
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Dec 18, 2019·Scientific Reports·Mariano MaffeiPaul A Heppenstall
Jul 28, 2011·Chembiochem : a European Journal of Chemical Biology·Xiaoli SunIvan R Corrêa
Mar 12, 2010·ACS Chemical Biology·Damien MaurelKai Johnsson
Jul 2, 2010·Analytical Chemistry·Mako Kamiya, Kai Johnsson
Jun 25, 2021·Chemical Science·Shahi Imam RejaKazuya Kikuchi

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