Broadening the scope of sortagging

Abstract

Sortases are enzymes occurring in the cell wall of Gram-positive bacteria. Sortase A (SrtA), the best studied sortase class, plays a key role in anchoring surface proteins with the recognition sequence LPXTG covalently to oligoglycine units of the bacterial cell wall. This unique transpeptidase activity renders SrtA attractive for various purposes and motivated researchers to study multiple in vivo and in vitro ligations in the last decades. This ligation technique is known as sortase-mediated ligation (SML) or sortagging and developed to a frequently used method in basic research. The advantages are manifold: extremely high substrate specificity, simple access to substrates and enzyme, robust nature and easy handling of sortase A. In addition to the ligation of two proteins or peptides, early studies already included at least one artificial (peptide equipped) substrate into sortagging reactions - which demonstrates the versatility and broad applicability of SML. Thus, SML is not only a biology-related technique, but has found prominence as a major interdisciplinary research tool. In this review, we provide an overview about the use of sortase A in interdisciplinary research, mainly for protein modification, synthesis of protein-polymer conjugates and immobilization of proteins on surfaces.

Scheme 1. Catalytic cycle of sortase-mediated ligation involving the formation of the thioester intermediate followed by attack of the oligoglycine nucleophile which finally results in the formation of the ligation product. Reprinted from ref. 11, Copyright (2016), with permission from Elsevier.

Scheme 2. Labeling (left) as well as circularization (right) of a protein with both peptide motifs for SML. Reprinted with permission from ref. 64.

Scheme 3. Functional small molecules equipped with the recognition sequence LPETG are recognized by native SrtA and covalently linked to the S. aureus cell wall. Reprinted with permission from ref. 91. Copyright (2010) American Chemical Society.

Scheme 4. SML of a protein of interest and a GGG-PEG-lipid modified cell membrane. Reprinted with permission from ref. 99.

Scheme 5. Overview of the principle to monitor cell–cell interactions: the acceptor cell is labeled by SML when ligand and receptor of two cell types interact. Reprinted by permission from Springer Nature from ref. 103, Copyright (2018).

Scheme 6. (a) Protein attachment on oligoglycine-functionalized liposomes. Reprinted with permission from ref. 108. Copyright (2012) American Chemical Society. (b) “Prebinding” strategy to enhance the efficiency of sortagging of proteins to liposomes. Reprinted with permission from ref. 113. Copyright (2017) American Chemical Society.

Scheme 7. SrtA catalysis applied for the synthesis of GPI–protein conjugates. Reprinted with permission from ref. 117. Copyright (2010) American Chemical Society.

Scheme 8. Strategy to equip both termini of one protein with labels using SrtA_pyogenes (SrtA_strep) and SrtA_aureus (Δ59-SrtA_staph). Reprinted with permission from ref. 121; https://pubs.acs.org/doi/abs/10.1021/ja902681k; further permissions related to the material excerpted should be directed to the ACS.

Scheme 9. Strategy from Warden-Rothman et al. for C-terminal labeling of a protein. Reprinted with permission from ref. 123. Copyright (2013) American Chemical Society.

Scheme 10. Strategy of preparing unnatural N-to-N and C-to-C fusion proteins. Reprinted from ref. 139.

Scheme 11. Two strategies for generating antibody–drug conjugates: SML and SPAAC (A), only SML (B). Reprinted without change from ref. 157, CC BY 4.0, https://www.mdpi.com/1422-0067/18/11/2284.

Scheme 12. (a) Sortagging with amine nucleophiles being unbranched at the α-carbon atom. Reprinted with permission from ref. 170. Copyright (2016) American Chemical Society. (b) Hydrazinolysis of proteins. Reprinted with permission from ref. 171.

Scheme 13. (a) The synthetic route to obtain a GFP-poly(OEGMA) conjugate via SML and grafting-from polymerization; (b) formation of GFP-polymer conjugates by combination of SML and SPAAC. Reprinted with permission from ref. 196 and 197.

Scheme 14. (a) Synthesis of eGFP-PEG conjugate via SML; (b) SDS-PAGE of the protein starting material and the product. Reprinted with permission from ref. 201. Copyright (2016) American Chemical Society.

Scheme 15. Immobilization of recombinant human thrombomodulin on glass slides via SML. Reprinted with permission from ref. 206. Copyright (2012) American Chemical Society.

Scheme 16. (a) Immobilization of LPETG-equipped-GFP onto GGG-functionalized gold surface via SML. (b) Strategy of immobilization two protein layers onto a gold surface by using the two different variants SrtA_pyogenes (Sp-Srt) and SrtA_aureus (Sa-Srt). Reprinted from ref. 210, Copyright (2015), with permission from Elsevier.

Scheme 17. Reaction cycle of immobilizing protein-LPETG on a pentaglycine-equipped surface and its removal by adding eSrtA together with an excess GGG. Reprinted without change from ref. 218, CC BY 4.0, https://www.nature.com/articles/ncomms11140.

Scheme 18. Ligation process of scFv-LPETG antibodies to eGFP (a), magnetic iron oxide particles (b), cells (c) and protein micelles (d). (a–c) Reprinted from ref. 222, https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.111.249375. (d) Reprinted with permission from ref. 225.

Scheme 19. Synthesis of a PNVCL microgel followed by a sortase-mediated immobilization of eGFP. Reprinted with permission from ref. 231. Copyright (2017) American Chemical Society.

Scheme 20. Crosslinking of peptide-equiped biopolymers to hydrogels by sortase A. The catalytic cycle is shown over the arrow. Reprinted from ref. 234, Copyright (2018), with permission from Elsevier.

Scheme 21. Overview of the functionalization of silica nanoparticles, PEG and PNIPAM polymer blocks with peptide sequences and the subsequent formation of NP–NP, NP–polymer and polymer–polymer structures catalyzed by SrtA. Reprinted from ref. 237.

Xiaolin Dai

Alexander Böker

Ulrich Glebe