Establishing strategies for sortase-catalyzed multi-peptide assemblies to profile T-cell selectivity

The bacterial transpeptidase sortase A has become a versatile tool for protein chemistry by catalyzing chemoselective ligation reactions of peptides, proteins and synthetic probes. During catalysis sortase cleaves a so-called sorting motif consisting of the amino acid sequence LPxTG or LPxTA at the threonine residue under concomitant formation of a sortase-bound thioester. The enzyme is deacylated by ligating the thioester to the glycine residue of a second peptide that serves as nucleophile.

While sortase A of S. aureus is widely used in biochemistry and chemical biology the assembly of multiple peptides is limited by the fact that each sorting motif and nucleophile is recognized by sortase, thereby leading to fragment shuffling and polymerization as a manifesting side reaction when fragment numbers increase. We plan to address these problems by establishing novel ligation sites to enable multi-fragment assemblies. We will use directed evolution methods to engineer sortase selectivity and activity. We plan to use these new tools for establishing tailor-made probes for immunology, in particular for the investigation of T-cell activation. 

Prof. Dr. Christian Freund
Freie Universität Berlin / Institut für Chemie und Biochemie

Tel. 030-838-51187
Fax. 030-838-56413

Email Prof. Freund


Prof. Dr. Dirk Schwarzer
Eberhard Karls Universität Tübingen

Tel.: +49 7071 29 - 73344
Fax: +49 7071 29 - 4815

Email Prof. Schwarzer

Publications within the SPP 1623 project

J. Bierlmeier, M. Alvaro-Benito, M. Scheffler, K. Sturm, L. Rehkopf, C. Freund, D. Schwarzer
Angew Chem Int Ed 2021 Nov 4. doi: 10.1002/anie.202109032. Online ahead of print. PMID: 34735044  
Link to the article

C. Freund, D. Schwarzer
Chembiochem2021, 22, 1347-1356
Engineered Sortases in Peptide and Protein Chemistry
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L. Schmohl, J. Bierlmeier, N. von Kügelgen, L. Kurz, P. Reis, F. Barthels, P. Mach, M. Schutkowski, C. Freund, D. Schwarzer
Bioorg Med. Chem. 2017, 25, 5002-5007
Identification of sortase substrates by specificity profiling
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L. Schmohl, J. Bierlmeier, F. Gerth, C. Freund, D. Schwarzer
J Pept Sci.2017, 23, 631-635
Engineering sortase A by screening a second-generation library using phage display
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W. Fischle, D. Schwarzer
ACS Chemical Biology 2016, 11(3), 689-705 (Review)
Probing Chromatin-modifying enzymes with Chemical Tools.
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A. Stützer, S. Liokatis, A. Kiesel, D. Schwarzer, R. Sprangers, J. Söding, P. Selenko, W. Fischle
Molecular Cell 2016, 61, 247-259
Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.
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A. Dose, J. Sindlinger, J. Bierlmeier, A. Bakirbas, K. Schulze-Osthoff, S. Einsele-Scholz, M. Hartl, F. Essmann, I. Finkemeier, D. Schwarzer
Angew. Chem. Int. Ed. 2016, 55, 1192-1195
Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes.
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J. Seidel, C. Klockenbusch, D. Schwarzer
Chembiochem 2016, 17(5), 398-402
Investigating deformylase and deacylase activity of mammalian and bacterial sirtuins.
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