Development of an Indole-Based Chemically Cleavable Linker Concept for Immobilizing Bait Compounds for Protein Pull-Down Experiments

作者：Sturm Martin; Leitner Alexander^*; Lindner Wolfgang

来源：Bioconjugate Chemistry, 2011, 22(2): 211-217.

DOI：10.1021/bc100330a

A novel linker chemistry based on a malondialdehyde-indole condensation reaction has been developed for the affinity-independent elution of targeted protein pull-downs. Previously developed in our lab for the tagging of tryptophan residues on proteins or peptides, the concept was extended for the design of a chemically cleavable linker system. Target molecules for interaction studies are immobilized on a solid support including the linker scaffold, and a typical pull-down experiment is carried out. After purification, the linker is cleaved by incubation with 50 mM pyrrolidine. A specific tyrosine kinase inhibitor, bosutinib, was coupled to agarose and acrylamide beads, respectively, via the new linker system, and a protein pull down experiment of putative interaction partners from a K562 whole cell lysate was performed. The system was found to be compatible with targeted protein pull downs, during the cleavage step, no protein hydrolysis or any degradation of amino acid side chains was apparent. From the pull down experiment, key targets of bosutinib such as the tyrosine kinase, Btk, were identified by liquid chromatography-tandem mass spectrometry.

• 出版日期2011-2