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Chembiochem. 2013 Nov 25;14(17):2310-21. doi: 10.1002/cbic.201300332. Epub 2013 Oct 22.

Towards functional orthogonalisation of protein complexes: individualisation of GroEL monomers leads to distinct quasihomogeneous single rings.

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Department for Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058 Basel (Switzerland); Current address: Department of Chemistry, Columbia University, 550 West 120th Street, New York, NY 10027 (USA).


The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single-ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.


biotechnology; molecular evolution; protein engineering; protein folding; protein-protein interactions; synthetic biology

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