Enzyme-Mediated Ligation Methods [1st ed.] 978-1-4939-9545-5;978-1-4939-9546-2

Table of contents :
Front Matter ....Pages i-xi
Sortase A Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody–Drug Conjugates (Rémy Gébleux, Manfred Briendl, Ulf Grawunder, Roger R. Beerli)....Pages 1-13
Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL) (Hejia Henry Wang, Andrew Tsourkas)....Pages 15-28
Cyclizing Disulfide-Rich Peptides Using Sortase A (Akello J. Agwa, David J. Craik, Christina I. Schroeder)....Pages 29-41
Chemoenzymatic Synthesis of Linear- and Head-to-Tail Cyclic Peptides Using Omniligase-1 (Marcel Schmidt, Timo Nuijens)....Pages 43-61
Site-Specific Labeling of Proteins Using the Formylglycine-Generating Enzyme (FGE) (Igor Rupniewski, David Rabuka)....Pages 63-81
Butelase 1-Mediated Ligation of Peptides and Proteins (Xinya Hemu, Xiaohong Zhang, Xiaobao Bi, Chuan-Fa Liu, James P. Tam)....Pages 83-109
Trypsiligase-Catalyzed Peptide and Protein Ligation (Sandra Liebscher, Frank Bordusa)....Pages 111-133
Site-Specific Antibody–Drug Conjugation Using Microbial Transglutaminase (Stephan Dickgiesser, Lukas Deweid, Roland Kellner, Harald Kolmar, Nicolas Rasche)....Pages 135-149
Tailoring Activity and Selectivity of Microbial Transglutaminase (Lukas Deweid, Olga Avrutina, Harald Kolmar)....Pages 151-169
SpyLigase-Catalyzed Modification of Antibodies (Vanessa Siegmund, Birgit Piater, Frank Fischer, Harald Kolmar)....Pages 171-192
Peptide Cyclization Catalyzed by Cyanobactin Macrocyclases (Wael E. Houssen)....Pages 193-210
In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis (Karen S. Harris, Simon Poon, Pedro Quimbar, Marilyn A. Anderson)....Pages 211-235
Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation (Jan Grünewald, Ansgar Brock, Bernhard H. Geierstanger)....Pages 237-278
Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization (Joseph G. Plaks, Joel L. Kaar)....Pages 279-297
BioID as a Tool for Protein-Proximity Labeling in Living Cells (Rhiannon M. Sears, Danielle G. May, Kyle J. Roux)....Pages 299-313
N-Myristoyl Transferase (NMT)-Catalyzed Labeling of Bacterial Proteins for Imaging in Fixed and Live Cells (Samuel H. Ho, David A. Tirrell)....Pages 315-326
Tubulin Tyrosine Ligase-Mediated Modification of Proteins (Marcus Gerlach, Tina Stoschek, Heinrich Leonhardt, Christian P. R. Hackenberger, Dominik Schumacher, Jonas Helma)....Pages 327-355
Inducible, Selective Labeling of Proteins via Enzymatic Oxidation of Tyrosine (Jorick J. Bruins, Criss van de Wouw, Jordi F. Keijzer, Bauke Albada, Floris L. van Delft)....Pages 357-368
Back Matter ....Pages 369-371

Citation preview

Methods in Molecular Biology 2012

Timo Nuijens Marcel Schmidt Editors

Enzyme-Mediated Ligation Methods

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Enzyme-Mediated Ligation Methods Edited by

Timo Nuijens and Marcel Schmidt EnzyPep B.V., Geleen, The Netherlands

Editors Timo Nuijens EnzyPep B.V. Geleen, The Netherlands

Marcel Schmidt EnzyPep B.V. Geleen, The Netherlands

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9545-5 ISBN 978-1-4939-9546-2 (eBook) https://doi.org/10.1007/978-1-4939-9546-2 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface The increasing understanding of biological systems provides exciting novel opportunities for their modulation using a wealth of therapeutic modalities. Whereas drug discovery has traditionally focused on small-molecule drugs, the advances in life science over the past 30 years have led to a significant expansion of this focus toward larger molecules beyond Lipinski’s rule of five, such as peptide or protein-based therapeutics. The emergence of this class of larger biological macromolecules as successful therapeutics has been accompanied by a complementary need of methodologies to enable their synthesis, derivatives thereof (e.g., containing unnatural moieties), or hybrid-modality conjugates (e.g., antibody-drug conjugates). Whereas initial efforts to modify peptides and proteins mostly focused on traditional chemical ligation methodologies such as native chemical ligation, alkyne-azide cycloaddition (“click” chemistry), and others, harnessing the biocatalytic power of nature’s most diverse toolkit, namely, enzymes, has not been in the center of focus in the beginning. Although enzymes had been widely used for the derivatization of small molecules, their use on peptide and protein level still had to be explored. However, over the past years, enzymemediated ligation strategies for the efficient preparation of modified peptides or proteins have been widely recognized as a powerful adjunct to existing chemical methodologies. Consequently, this increased interest resulted in the discovery of novel naturally occurring enzymes and the creation of optimized variants thereof. The available set of ligases comprises enzymes from various classes such as engineered proteases (e.g. subtilisin variants), transpeptidases (sortases), or lipid transferases (e.g., PPTase). Current applications include the use in linear peptide synthesis, in peptide macrocyclization, and in peptide and protein (e.g., antibody) derivatization, as a tool both in research and in manufacture. In either environment, the use of enzymes in many cases enables site-specific, efficient ligations under mild conditions and provides an elegant link between chemistry and biology. In this edition of Methods in Molecular Biology, we cover many different enzymecatalyzed ligation methodologies, and the book is devoted to those students and scientists from different disciplines, who consider to use enzymatic strategies for a variety of research questions. We hope to facilitate the use of enzymes by providing protocols for a variety of chemical transformations and to break the “virtual barrier” for many people to actively and widely use enzymes as a tool in academic research as well as in industrial settings. Geleen, The Netherlands

Timo Nuijens Marcel Schmidt

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Sortase A Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody–Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Re´my Ge´bleux, Manfred Briendl, Ulf Grawunder, and Roger R. Beerli 2 Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL) . . . . . . . . . . . . . . . . . . . . Hejia Henry Wang and Andrew Tsourkas 3 Cyclizing Disulfide-Rich Peptides Using Sortase A . . . . . . . . . . . . . . . . . . . . . . . . . . Akello J. Agwa, David J. Craik, and Christina I. Schroeder 4 Chemoenzymatic Synthesis of Linear- and Head-to-Tail Cyclic Peptides Using Omniligase-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marcel Schmidt and Timo Nuijens 5 Site-Specific Labeling of Proteins Using the Formylglycine-Generating Enzyme (FGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Igor Rupniewski and David Rabuka 6 Butelase 1-Mediated Ligation of Peptides and Proteins . . . . . . . . . . . . . . . . . . . . . . Xinya Hemu, Xiaohong Zhang, Xiaobao Bi, Chuan-Fa Liu, and James P. Tam 7 Trypsiligase-Catalyzed Peptide and Protein Ligation . . . . . . . . . . . . . . . . . . . . . . . . Sandra Liebscher and Frank Bordusa 8 Site-Specific Antibody–Drug Conjugation Using Microbial Transglutaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephan Dickgiesser, Lukas Deweid, Roland Kellner, Harald Kolmar, and Nicolas Rasche 9 Tailoring Activity and Selectivity of Microbial Transglutaminase . . . . . . . . . . . . . . Lukas Deweid, Olga Avrutina, and Harald Kolmar 10 SpyLigase-Catalyzed Modification of Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . Vanessa Siegmund, Birgit Piater, Frank Fischer, and Harald Kolmar 11 Peptide Cyclization Catalyzed by Cyanobactin Macrocyclases . . . . . . . . . . . . . . . . Wael E. Houssen 12 In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis . . . . . . . . . . . . . . . Karen S. Harris, Simon Poon, Pedro Quimbar, and Marilyn A. Anderson 13 Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ¨ newald, Ansgar Brock, and Bernhard H. Geierstanger Jan Gru 14 Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joseph G. Plaks and Joel L. Kaar

vii

v ix

1

15 29

43

63 83

111

135

151 171 193

211

237

279

viii

15

Contents

BioID as a Tool for Protein-Proximity Labeling in Living Cells. . . . . . . . . . . . . . . Rhiannon M. Sears, Danielle G. May, and Kyle J. Roux N-Myristoyl Transferase (NMT)-Catalyzed Labeling of Bacterial Proteins for Imaging in Fixed and Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samuel H. Ho and David A. Tirrell Tubulin Tyrosine Ligase-Mediated Modification of Proteins . . . . . . . . . . . . . . . . . Marcus Gerlach, Tina Stoschek, Heinrich Leonhardt, Christian P. R. Hackenberger, Dominik Schumacher, and Jonas Helma Inducible, Selective Labeling of Proteins via Enzymatic Oxidation of Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jorick J. Bruins, Criss van de Wouw, Jordi F. Keijzer, Bauke Albada, and Floris L. van Delft

299

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

369

16

17

18

315 327

357

Contributors AKELLO J. AGWA  Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia BAUKE ALBADA  Laboratory of Organic Chemistry, Wageningen University and Research, Wageningen, The Netherlands MARILYN A. ANDERSON  Hexima Limited, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia OLGA AVRUTINA  Clemens-Scho¨pf Institute for Organic Chemistry and Biochemistry, Technische Universit€ a t Darmstadt, Darmstadt, Germany ROGER R. BEERLI  NBE-Therapeutics Ltd., Basel, Switzerland XIAOBAO BI  School of Biological Sciences, Nanyang Technological University, Singapore, Singapore FRANK BORDUSA  Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein Center, Martin-Luther-University Halle-Wittenberg, Halle, Germany MANFRED BRIENDL  NBE-Therapeutics Ltd., Basel, Switzerland ANSGAR BROCK  Biotherapeutics, Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA JORICK J. BRUINS  Laboratory of Organic Chemistry, Wageningen University and Research, Wageningen, The Netherlands DAVID J. CRAIK  Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia LUKAS DEWEID  Clemens-Scho¨pf Institute for Organic Chemistry and Biochemistry, Technische Universit€ a t Darmstadt, Darmstadt, Germany STEPHAN DICKGIESSER  ADCs & Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany FRANK FISCHER  Biomolecule Analytics, Merck KGaA, Darmstadt, Germany RE´MY GE´BLEUX  NBE-Therapeutics Ltd., Basel, Switzerland BERNHARD H. GEIERSTANGER  Biotherapeutics, Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA MARCUS GERLACH  Department of Biology II, LMU Munich, Planegg/Martinsried, Germany JAN GRU¨NEWALD  Biotherapeutics, Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA ULF GRAWUNDER  NBE-Therapeutics Ltd., Basel, Switzerland CHRISTIAN P. R. HACKENBERGER  Department of Chemical-Biology, Leibniz-Institut fu¨r Molekulare Pharmakologie (FMP), Berlin, Germany; Department of Chemistry, Humboldt Universit€ at zu Berlin, Berlin, Germany KAREN S. HARRIS  Hexima Limited, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia JONAS HELMA  Department of Biology II, LMU Munich, Planegg/Martinsried, Germany XINYA HEMU  School of Biological Sciences, Nanyang Technological University, Singapore, Singapore SAMUEL H. HO  Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

ix

x

Contributors

WAEL E. HOUSSEN  Marine Biodiscovery Centre, Chemistry Department, University of Aberdeen, Aberdeen, UK; Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK; Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt JOEL L. KAAR  Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA JORDI F. KEIJZER  Laboratory of Organic Chemistry, Wageningen University and Research, Wageningen, The Netherlands ROLAND KELLNER  ADCs & Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany HARALD KOLMAR  Clemens-Scho¨pf Institute for Organic Chemistry and Biochemistry, Technische Universit€ a t Darmstadt, Darmstadt, Germany HEINRICH LEONHARDT  Department of Biology II, LMU Munich, Planegg/Martinsried, Germany SANDRA LIEBSCHER  Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein Center, Martin-Luther-University Halle-Wittenberg, Halle, Germany CHUAN-FA LIU  School of Biological Sciences, Nanyang Technological University, Singapore, Singapore DANIELLE G. MAY  Enabling Technology Group, Sanford Research, Sioux Falls, SD, USA TIMO NUIJENS  EnzyPep B.V., Geleen, The Netherlands BIRGIT PIATER  ADCs & Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany JOSEPH G. PLAKS  Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA SIMON POON  Hexima Limited, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia PEDRO QUIMBAR  Hexima Limited, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia DAVID RABUKA  Catalent Pharma Solutions, Richmond, CA, USA NICOLAS RASCHE  ADCs & Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany KYLE J. ROUX  Enabling Technology Group, Sanford Research, Sioux Falls, SD, USA; Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA IGOR RUPNIEWSKI  Catalent Pharma Solutions, Richmond, CA, USA MARCEL SCHMIDT  EnzyPep B.V., Geleen, The Netherlands; Van’t Hoff Institute of Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands CHRISTINA I. SCHROEDER  Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia DOMINIK SCHUMACHER  Department of Biology II, LMU Munich, Planegg/Martinsried, Germany; Department of Chemical-Biology, Leibniz-Institut fu¨r Molekulare Pharmakologie (FMP), Berlin, Germany RHIANNON M. SEARS  Enabling Technology Group, Sanford Research, Sioux Falls, SD, USA; Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA VANESSA SIEGMUND  Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt, Germany TINA STOSCHEK  Department of Biology II, LMU Munich, Planegg/Martinsried, Germany

Contributors

xi

JAMES P. TAM  School of Biological Sciences, Nanyang Technological University, Singapore, Singapore DAVID A. TIRRELL  Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA ANDREW TSOURKAS  Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA CRISS VAN DE WOUW  Laboratory of Organic Chemistry, Wageningen University and Research, Wageningen, The Netherlands FLORIS L. VAN DELFT  Laboratory of Organic Chemistry, Wageningen University and Research, Wageningen, The Netherlands HEJIA HENRY WANG  Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA XIAOHONG ZHANG  School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Chapter 1 Sortase A Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody–Drug Conjugates Re´my Ge´bleux, Manfred Briendl, Ulf Grawunder, and Roger R. Beerli Abstract Antibody–drug conjugates (ADCs) are highly potent targeted anticancer therapies. They rely on the linking of a selectively targeting antibody moiety with potent cytotoxic payloads to effect antitumoral activity. In recent years, one focus in the ADC field was to create novel methods for site-specifically conjugating payloads to antibodies. The method presented here is based on the S. aureus sortase A-mediated transpeptidation reaction. This method requires antibodies to be engineered in such a way that they possess the sortase recognition pentapeptide motif LPETG on the C-terminus of the immunoglobulin heavy and/or light chains. In addition, the toxin must contain an oligoglycine motif in order to make it a suitable substrate for sortase A. Here we describe a detailed method to conjugate a pentaglycine-modified toxin to the C-termini of LPETG-tagged antibody heavy and light chains using sortase-mediated antibody conjugation (SMAC-Technology™). Highly homogenous, site-specifically conjugated ADCs with controlled drug to antibody ratio and improved overall properties can be obtained with this method. Key words Antibody–drug conjugate, ADC, Sortase A-mediated ligation, Transpeptidation, Sitespecific conjugation, Sortase-mediated antibody conjugation, SMAC, Anthracycline, Maytansine, Auristatin

1

Introduction Site-specific conjugation of toxins to antibodies has made a tremendous impact on the field of antibody–drug conjugates (ADCs). Classical methods for ADC production include chemical conjugation via cysteine or lysine amino acid side chains, which often results in heterogeneous mixtures of ADCs where the drug-to-antibody ratio (DAR) and the site of conjugation cannot be precisely controlled. Therefore, these heterogeneously conjugated ADCs were associated with variable pharmacokinetics, efficacy, safety and stability properties. Today, various methods for site-specific conjugation have been developed in order to produce homogenous ADCs with a predetermined DAR and enhanced overall properties. These methods include engineering of cysteines, incorporation of

Timo Nuijens and Marcel Schmidt (eds.), Enzyme-Mediated Ligation Methods, Methods in Molecular Biology, vol. 2012, https://doi.org/10.1007/978-1-4939-9546-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

1

2

Re´my Ge´bleux et al.

unnatural amino acids used as specific chemical handles for conjugation, glycan engineering and enzyme assisted ligation [1–6]. Especially, enzyme-assisted ligation methodologies have gained increased interest in the last years. The method described hereafter, sortase-mediated antibody conjugation (SMAC™), relies on a platform based on the S. aureus sortase A-mediated transpeptidation reaction. Similar “sortagging” methods have been used in the past to perform protein-protein ligation [7, 8], label protein with dyes [9–11] and conjugate small peptides or dyes to antibody fragments [12–14]. In the method described in this chapter, the antibody and the toxin used in the reaction need to be slightly modified such that they become compatible with the enzyme substrate requirements (see Fig. 1). As described previously [15, 16], the antibody must be Cterminally tagged with a sortase A recognition motif (LPETG) and optionally contains a TwinStrep tag (underlined below) for subsequent removal of underconjugated ADC. The resulting tag sequences are GGGGS-LPETGG-WSHPQFEK-GGGSGGGSGGSSAWSHPQFEK-GS for the IgL chain (LC) and LPETGGWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK-GS for the IgH chain (HC). C-terminal conjugation has no impact on antigen binding as it is distant from the complementarity determining regions (CDRs) that drive antigen recognition. In addition to the antibody, the toxin needs to be modified with a pentaglycine peptide (such as in Fig. 2 (in blue)) to become an acceptable substrate for the enzyme. The conjugation process is carried out in a one-pot reaction combining the C-terminally LPETG-tagged antibody, the pentaglycinemodified toxin and the sortase A enzyme, which may be wild-type

Fig. 1 Schematic representation of sortase A-mediated antibody conjugation (SMAC™). Transpeptidation mechanism is initiated by the nucleophilic attack of the thiol group of C184 of sortase A to the peptide bond between the threonine and the glycine of the LPETG motif, yielding a covalent thioacyl intermediate and the release of the terminal glycine and additional C-terminal sequences if present. The thioacyl can then be resolved by the nucleophilic attack of an oligoglycine stretch (such as G5-Toxin), resulting in the formation of a new peptide bond between the threonine and the incoming glycine (LPETG5-Toxin)

Sortase A Conjugated ADCs

3

Fig. 2 Chemical Structure of various linker payload constructs comprising a toxin derivative and a pentaglycine peptide combined with certain linkers. (a) Gly5-EDA-SMCC-DM1. (b) Gly5-EDA-PNU. (c) Gly5-vc-PAB-MMAE

sortase A [17, 18], or an evolved sortase A variant with improved enzymatic properties (eSrtA) [15, 19]. The reaction mixture is incubated at 20
C or room temperature for a few hours. Subsequently, the desired ADC can be purified from the reaction mixture, which in a lab-scale process can easily be accomplished, for example, by purification via Protein A chromatography. Afterward, the resulting ADC is formulated in an appropriate buffer. Optional steps to improve the final ADC product include DAR-enrichment and endotoxin removal for use in in vivo experiments. DAR determination by reversed-phase high-performance liquid chromatography (RP-HPLC) is the most common analysis performed on the purified ADC.

2

Materials Prepare all solutions using Ultrapure Water (by purifying deionized water so that it reaches a resistivity of 18.2 MΩ cm at 25
C) and analytical grade reagents. If the ADCs are intended for in vivo use, use endotoxin-free containers and reagents. All toxin contaminated material and solutions must be disposed of according to local regulations as toxic waste. A centrifuge compatible with 15 mL Falcon tubes is required for this method.

4

Re´my Ge´bleux et al.

2.1 SortaseMediated Antibody Conjugation (SMAC)

1. 5 SMAC buffer: 250 mM HEPES, 5 mM CaCl2, 50% glycerol (v/v), pH 7.5 (see Note 1). 2. 5 M NaCl solution. 3. Solution of LPETG-TwinStrep C-terminally tagged antibodies with a concentration of at least 20 μM (Produced according to [6, 7]). 4. 1 mM stock solution of pentaglycine-modified toxin in water (see Note 2). 5. Solution of evolved sortase A enzyme (eSrtA) with a concentration of at least 50 μM (produced according to [6, 7]). Many eSrtA variants are also available from commercial vendors.

2.2 Purification of the SMAC Reaction Using a Protein A Column

1. Gravity flow Protein A column such as rProtein A GraviTrap columns (GE Healthcare, #28-9852-54, 1 mL resin, suitable for 12–15 mg ADC). 2. Protein A Equilibration/Wash buffer: 25 mM HEPES, 150 mM NaCl, 10% glycerol (v/v), pH 7.5. 3. Protein A Elution buffer: 100 mM glycine, 50 mM NaCl, 10% glycerol (v/v), pH 2.7. 4. Protein A Neutralization buffer: 1 M HEPES, pH 8.0. 5. Protein A Regeneration buffer: 1.5 M NaSCN in water. 6. Protein A Storage buffer: 25 mM HEPES, 150 mM NaCl, 10% glycerol (v/v), pH 7.5, 0.2% sodium azide.

2.3 StrepTactin Enrichment (Optional)

1. Gravity flow Strep-Tactin®XT Superflow® column (IBA, 1 mL, # 2-4012-001). 2. Buffer W: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0. 3. Buffer BXT: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 50 mM biotin, pH 8.0. 4. Regeneration buffer: 10 mM NaOH.

2.4

Formulation

1. Phosphate buffered saline (PBS) or other buffer of choice. 2. Desalting/Formulation column such as Zeba Spin column (ThermoFisher, #89892, 5 mL resin, suitable for 0.5–2 mL sample). 3. 0.22 μm PVDF syringe filter unit and corresponding disposable syringe.

2.5 Endotoxin Removal (Optional)

1. Endotrap resin such as Pierce High Capacity Endotoxin Removal Resin (ThermoFisher, #88275, 0.5 mL columns, suitable for 1–4 mL sample). 2. Regeneration buffer: 0.2 M NaOH (in water or 95% ethanol).

Sortase A Conjugated ADCs

5

3. Rotating wheel (e.g., Stuart Rotator SB3 or similar). 4. 2 M NaCl solution. 2.6 RP-HPLC Analysis

1. HPLC system with appropriate UV-Vis detector (see Note 3). ˚ , 2.1  50 mm, 2. RP-HPLC column (such as PLRP-S 1000 A 5 μm (Agilent, #PL1912-1502) or MAbPac™ RP, 4 μM 3.0  100 mm (ThermoFisher, #088644)).

3. HPLC solution: 0.5 M DTT and 10 U/μL Fabricator/IdeS (Genovis). 4. Buffer A: Ultrapure water containing 0.1% Trifluoroacetic acid. 5. Buffer B: Acetonitrile containing 0.1% Trifluoroacetic acid. 6. Heating block. 7. Centrifuge for small tubes (0.5–2 mL).

3

Methods Carry out all procedures at 20
C or room temperature, unless otherwise specified.

3.1 SortaseMediated Antibody Conjugation (SMAC)

1. Mix all the components of the SMAC reaction in a 15 mL Falcon tube to reach the final concentrations as described in Table 1 (see Notes 4 and 5). It is recommended to add the components in the following order: H2O—SMAC buffer— NaCl—Toxin—Antibody—Sortase A. 2. Incubate for 4 h.

3.2 Protein A Purification of ADC

1. Centrifuge the SMAC reaction at 4,000  g for 10 min. 2. Place the protein A column on top of a 50 mL Falcon tube, open both ends of the column and let the storage buffer drip through the resin by gravity flow. 3. Equilibrate the column with at least 24 column volumes (CV) of Protein A Wash buffer. 4. Load the SMAC reaction onto the column and collect the flowthrough (FT) in a separate tube to be disposed of as toxic waste. 5. Wash the Protein A column with at least 36 CV of Protein A Wash buffer to remove excess eSrtA and toxin. 6. Add 5 CV of Elution buffer to the Protein A column and collect the ADC in 250 μL fractions in Eppendorf tubes. 7. Determine which fractions contain ADC using UV-Vis spectrophotometry (e.g., NanoDrop), Bradford assay, or any other suitable method.

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Re´my Ge´bleux et al.

Table 1 Composition of the SMAC reaction H2O to final SMAC buffer NaCl Toxin Antibody Sortase Final volume (μL) (fold) (mM) (μM) (μM) A (μM) volume (μL) Final concentration

/

1

Stock Concentration

/

5

150

200

5000 1000

10

4

7000

>20 μM

>50 μM /

8. Pool ADC containing fractions and neutralize with 1:15 to 1:25 volumes (v/v) of Protein A Neutralization buffer (see Note 6). 9. The Protein A column can be regenerated by washing with 5 CV of Protein A Elution buffer, 24 CV of Protein A Regeneration buffer, and 24 CV of Protein A Wash buffer. The regenerated column can be stored at 4
C in Protein A Storage buffer. 3.3 StrepTactin Enrichment (Optional)

The purpose of the StrepTactin chromatography step is the removal of underconjugated ADC in cases where conjugation efficiency is not satisfactory. 1. Further neutralize the ADC solution with 1:6 volumes (v/v) Neutralization buffer in order to reach a pH between 7.5 and 8.0, which is compatible with binding to the StrepTactin resin (see manufacturer’s instructions). 2. Place the StrepTactin column on top of a 50 mL tube. Remove the top cap of the column followed by the bottom cap and let the storage buffer drip through the resin by gravity flow. 3. Equilibrate the StrepTactin column with 2 mL of Buffer W. 4. Load the ADC onto the resin and collect the FT in 250 μL fractions in Eppendorf tubes (see Note 7). 5. Add 2 mL of buffer W onto the column in order to remove the residual ADC from the dead volume of the resin and fractionate the FT in 250 μL fractions in Eppendorf tubes. 6. Pool all the FT fractions which contain ADC. Use any suitable method to detect protein, such as UV/VISspectrophotometry or a Bradford assay. 7. The StrepTactin column can be regenerated by washing with 5 CV of buffer W, 3 CV of buffer BXT, 15 CV of Regeneration buffer and 10 CV of buffer W. The regenerated column can be stored at 4
C in buffer W containing 0.02% sodium azide.

Sortase A Conjugated ADCs

3.4

Formulation

7

Always loosen top cap during centrifugation step. Before starting with step 1, see Note 8. 1. Snap open the bottom closure of the desalting column, loosen the cap (but do not fully remove it) and place the column into a 15 mL Falcon tube. Centrifuge for 2 min at 1000  g to remove the storage solution. Discard the FT. 2. Place the column into the same Falcon tube, remove the cap and add 2.5 mL of PBS (or the formulation buffer of choice) on top of the resin. Put the cap back loosely onto the column (do not tighten) and centrifuge for 2 min at 1000  g. Discard the FT. Repeat step 2 four times (five washes in total). 3. Place the column into a clean 15 mL Falcon, remove the cap and add the ADC solution on top of the resin (see Note 9). Put the cap back loosely on the column and centrifuge for 3 min at 1000  g. 4. Sterile filter the ADC with a 0.22 μm syringe filter unit and corresponding disposable syringe (see Note 10). 5. Measure the concentration of the ADC using a suitable method (UV-VIS, Bradford, HPLC-MS) (see Note 11). 6. Prepare ADC aliquots of the desired volume and snap-freeze in liquid nitrogen. Store frozen aliquots at 80
C until further use (see Note 12).

3.5 Endotoxin Removal (Optional)

Sortase enzymes are typically produced in bacteria and are likely to contain significant quantities of endotoxins. Similarly, many linkerpayload precursors are produced by fermentation and may contain endotoxins. While the endotoxin removal step can be omitted for most in vitro applications of ADCs, endotoxin removal is strongly advised if the ADC is to be used in in vivo studies. Always remove bottom cap and loosen top cap during centrifugation step. Always put back bottom cap after centrifugation and before addition of solution to the resin. Always have both bottom and top cap tightened on the column during resuspension or incubation step. 1. Equilibrate the endotoxin removal column to room temperature. Snap open the bottom closure. Place column into a 15 mL Falcon tube. Centrifuge for 1 min at 500  g to remove storage solution. Discard FT. 2. Regenerate column for 2 h with 3.5 mL of 0.2 M NaOH in 95% EtOH or overnight with 3.5 mL of 0.2 M NaOH in water. Invert the column several times until the resin is suspended in the solution. Incubate for 2 h with slow complete inversion on a rotating wheel such that the resin is at all times in suspension. Centrifuge for 1 min at 500  g. Discard FT.

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3. Add 3.5 mL of 2 M NaCl solution. Invert the column several times until the resin is suspended in the solution. Centrifuge for 1 min at 500  g. Discard FT. 4. Add 3.5 mL of ultrapure water. Invert the column several times until the resin is suspended in the solution. Centrifuge for 1 min at 500  g. Discard FT. 5. Add 3.5 mL of PBS (or formulation buffer of choice). Invert the column several times until resin is suspended in the solution. Centrifuge for 1 min at 500  g. Discard FT. Repeat step 5 twice (total of three washes). 6. Add ADC sample. Incubate for 60 min with slow complete inversion on a rotating wheel such that the resin is in suspension at all times. Place the column in a new 15 mL Falcon. Centrifuge for 1 min at 500  g. Retain eluate. Discard column in the toxic waste. 3.6 RP-HPLC Analysis of ADCs

1. Prepare the HPLC system according to manufacturer’s instructions. Briefly, turn on the UV-Vis lamps and let them warm up for at least 1 h. Equilibrate the RP-column with 90% Buffer A/10% Buffer B for at least 60 min at 0.5 mL/min with a column temperature of 70
C (see Note 13). 2. Prepare a sufficient amount of ADC and parental mAb for analysis. Dilute the samples to 0.5 mg/mL containing either 10% 0.5 M DTT solution (for DAR determination on the LC) or 10% FabRICATOR/IdeS solution (for DAR determination on the HC) (see Note 14). 3. Incubate the samples for 30 min at 37
C. Centrifuge at 14,000  g for 10 min. Transfer the samples into appropriate glass vials for HPLC analysis. 4. Run the sample on the HPLC with the following buffer B gradient: 9 min from 25% to 40%, 2 min from 40% to 100%, 2 min at 100%, 2 min from 100% to 25% (see Note 15). 5. Overlay the traces of the ADC and its parental mAb. Annotate the chromatogram peaks from left to right as follows: For DTT treatment; unconjugated LC, conjugated LC, unconjugated HC, conjugated HC (see Fig. 3a). For IdeS treatment; unconjugated Fc/2, conjugated Fc/2, unconjugated F(ab0 )2, conjugated F(ab0 )2 (see Fig. 3b) (see Note 16). 6. Calculate the DAR on the LC using the area under the curve (AUC) values of the annotated unconjugated LC (uLCAUC) and conjugated LC (cLCAUC) from the DTT chromatogram with the following formula: DARLC ¼ 2  cLCAUC =ðcLCAUC þ uLCAUC Þ:

Sortase A Conjugated ADCs

9

Fig. 3 RP-HPLC spectra of anthracycline-conjugated ADCs based on trastuzumab. (a) Profile at 214 nm of ADC (in blue) and parental mAb (in black) following treatment by DTT. (b) Profile at 214 nm of ADC (in blue) and parental mAb (in black) following treatment by IdeS. (c) Overlaying traces of conjugated LC (DTT treatment) or conjugated Fc/2 (IdeS treatment) at 248 nm (protein, in blue) and 488 nm (toxin, in red). Arrows show the small difference in AUC indicating a slight underconjugation of the ADC

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7. Calculate the DAR on the HC using the AUC values of the annotated unconjugated Fc/2 (uFcAUC) and conjugated Fc/2 (cFCAUC) from the IdeS chromatogram with the following formula: DARHC ¼ 2  cFcAUC =ðcFcAUC þ uFcAUC Þ: 8. Calculate the total DAR of the ADC by adding the DARLC and DARHC together.

4

Notes 1. Calcium is a cofactor used by sortase A and is therefore mandatory for its activity. The use of glycerol is recommended to obtain better overall solubility of the SMAC components. The reaction can be performed at a wide range of pHs but with slower kinetics. Also, undesired side reactions can take place at nonoptimal pHs such as hydrolysis or cross-linking with the side chain of lysine residues [12]. 2. Most Gly5-modified toxins are water soluble. If the linkerpayload construct is not completely water soluble, it is recommended to dissolve the toxin in a small portion of organic solvent first (such as dimethylacetamide (DMA) or dimethylsulfoxide (DMSO)) before adding water. 3. It is recommended to detect the ADC at 214 or 280 nm. To confirm toxin conjugates, a wavelength at which the toxin has its maximal absorbance or where the toxin absorbs while the mAb does not can also be used. For example, in the case of anthracycline-based ADCs, the toxin can be detected in the visible range at 488 nm. 4. For some more hydrophobic drugs that are difficult to dissolve in water alone or difficult antibodies that are prone to precipitation, it is possible to include a small amount of organic solvent during the SMAC (i.e., 5–10% of DMA or DMSO) to improve the overall solubility of the toxin and the antibody. The organic solvent has an influence on the activity of sortase A, therefore it may be required to increase the final enzyme concentration up to 10 μM or increase the incubation time to reach comparable DAR values to those of a standard conjugation. 5. Adjust the final concentration of NaCl to 150 mM. Keep in mind that the antibody or the sortase may also contain some NaCl in their respective buffers. 6. Some ADCs are not suitable for neutralization. It is therefore recommended to test the neutralization buffer ratio to be used on a small portion of the pooled ADC product. For some extreme cases, where the neutralization is not feasible at all, it

Sortase A Conjugated ADCs

11

is possible to directly perform the formulation step (see Subheading 3.4). It is not recommended to store the ADC for extended time under acidic conditions. 7. This step is meant to remove partially underconjugated antibody fractions and is not an affinity capture. Therefore, it is essential to note that the desired ADC product will be in the FT. This step can also remove residual sortase if it also contains a TwinStrep tag. 8. If the pH of the formulation buffer is not between 6 and 8, the endotoxin removal step (Subheading 3.5) must be performed prior to the formulation step (Subheading 3.4) in order for the pH to be in a range compatible with the endotoxin resin (pH 6–8). If possible, it is better to remove the endotoxins as a final step, in order to avoid any contamination from the formulation buffer. The formulation column may not be completely endotoxin free even after extensive washing. 9. In case the volume of ADC solution is too small (8 h). Although with that amount of time, the reaction is very likely to go to nearcompletion, the hydrolysis side product will also accumulate and may be extremely difficult to separate from the desired ligation product.

Proximity-Based Sortase-Mediated Ligation

27

20. Resin postcapture (Lane 2) should have an additional band (compared to resin precapture, Lane 1) corresponding to the molecular weight of the target protein-LPETG-SpyTag plus that of SpyCatcher-SrtA-His6 (28.5 kDa), indicating successful capture of the target protein. If no such band can be detected, then no soluble target protein was expressed and expression conditions need to be further optimized. If there is no SpyCatcher-SrtA-His6 band—that is, there is only a large molecular weight target protein-LPETG-SpyTag-SpyCatcherSrtA-His6 band—then the resin has been fully saturated with target protein. Reuse the saved flow-through for additional rounds of purification. If there is a significant band corresponding to SpyTag-SpyCatcher-SrtA-His6 (31 kDa, runs ~3 kDa above SpyCatcher-SrtA-His6), then sufficient Ca2+ is present during the capture and wash steps to induce SrtA hydrolysis of the target protein off the resin. Doublecheck that all buffers have been prepared with ultrapure deionized water (18 MΩ resistance at 25
C). If that does not solve the issue, following centrifugation, resuspend the expression culture pellet in PBS + 10 mM EDTA, rotate at room temperature for 10 min, and repellet by centrifuging at 4000  g for 10 min. This removes most of the Ca2+ that accumulates on the E. coli during expression. The ligated product should be of high purity since no imidazole is present in the SrtA ligation buffer. Any contaminants are E. coli proteins nonspecifically bound to the cobalt resin that slowly leaches off during the ligation reaction. If purity is unsatisfactory, perform additional rounds of washes with the wash buffer prior to the ligation reaction. The intensity of the target protein-LPETG-SpyTag-SpyCatcher-SrtA-His6 band should be significantly diminished in the resin postligation sample (Lane 5), with a concomitant increase in the intensity of the SpyTag-SpyCatcher-SrtA-His6 band. If a substantial amount of target protein-LPETG-SpyTag-SpyCatcher-SrtA-His6 still remains, then the reaction has not progressed to completion and additional ligation buffer can be added to the resin. References 1. Krall N, da Crus FP, Boutureira O et al (2016) Site-specific protein-modification for basic biology and drug development. Nat Chem 8 (2):103–113 2. Stephanopoulos N, Francis M (2011) Choosing an effective protein bioconjugation strategy. Nat Chem Biol 7(12):876–884 3. Mazmanian S, Liu G, Ton-That H et al (1999) Staphylococcus aureus sortase, an enzyme that

anchors surface proteins to the cell wall. Science 285(5428):760–763 4. Mao H, Hart SA, Schink A et al (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126(9):2670–2671 5. Pishesha N, Ingram JR, Ploegh HL (2018) Sortase A: a model for transpeptidation and

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its biological applications. Annu Rev Cell Dev Biol 34:163–188 6. Popp M, Antos JM, Grotenbreg GM et al (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(13):707–708 7. Pritz S, Wolf Y, Kraetke O et al (2007) Synthesis of biologically active peptide nucleic acidpeptide conjugates by sortase-mediated ligation. J Org Chem 72(10):3909–3912 8. Parthasarathy R, Subramanian S, Boder ET (2007) Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug Chem 18(2):469–476 9. Chan L, Cross HF, She JK et al (2007) Covalent attachment of proteins to solid supports and surfaces via sortase-mediated ligation. PLoS One 2(11):e1164 10. Tsukiji S, Nagamune T (2009) Sortasemediated ligation: a gift from gram-positive bacteria to protein engineering. Chembiochem 10(5):787–798 11. Schmidt M, Toplak A, Quaedflieg PJ et al (2017) Enzyme-mediated ligation technologies for peptides and proteins. Curr Opin Chem Biol 38:1–7 12. Jacobitz A, Kattke MD, Wereszczynski J et al (2018) Sortase transpeptidase: structural biology and catalytic mechanism. In: Karabencheva-Christova T (ed) Structural and mechanistic enzymology, Advances in protein chemistry and structural biology, vol 109. Elsevier, Cambridge, pp 223–264

13. Heck T, Phu-Hu P, Yerlikaya A et al (2014) Sortase A catalyzed reaction pathways: a comparative study with six SrtA variants. Cat Sci Technol 4:2946–2956 14. Policarpo RL, Kang H, Liao X et al (2014) Flow-based enzyme ligation by sortase A. Angew Chem Int Ed 53(35):9203–9208 15. Frankel BA, Kruger RG, Robinson DE et al (2005) Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry 44(33):11188–11200 16. Warden-Rothman R, Caturegli I, Popik V et al (2013) Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal Chem 85(22):11090–11097 17. Wang HH, Altun B, New K et al (2017) Proximity-based sortase-mediated ligation. Angew Chem Int Ed 56(19):5349–5352 18. Zakeri B, Fierer JO, Celik E et al (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. Proc Natl Acad Sci U S A 109(12): e690–e697 19. Li L, Fierer JO, Rapoport TA et al (2014) Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag. J Mol Biol 426(2):309–317 20. Studier F (2005) Protein production by autoinduction in high-density shaking cultures. Protein Expr Purif 41(1):207–234

Chapter 3 Cyclizing Disulfide-Rich Peptides Using Sortase A Akello J. Agwa, David J. Craik, and Christina I. Schroeder Abstract Sortase A (SrtA) is an enzyme obtained from Staphylococcus aureus that catalyzes site-specific transpeptidation of surface proteins to the bacterial cell membrane. SrtA recognizes an LPXTG amino acid motif and cleaves between the Thr and Gly to form a thioester-linked acyl–enzyme intermediate. The intermediate is resolved in the presence of a nucleophilic N-terminal polyglycine resulting in ligation of the acyl donor to the polyglycine acceptor. Here we describe the application of SrtA as a tool for the cyclization of disulfiderich peptides. Reactions are typically tailored to each disulfide-rich peptide with optimal conditions producing yields of 40–50% cyclized peptide. Key words Cyclotide, Cyclization, Kalata B1, Macrocycle, Peptide ligation, Semienzymatic, Head-totail cyclic peptide, Sortase A

1

Introduction Disulfide-rich peptides are obtainable from a wide variety of plant and animal species and typically adopt well-defined and stable three-dimensional structures. Cyclotides are an example of disulfide-rich macrocycles that are distinguished by their knotted core enclosed within a head-to-tail cyclized backbone. These ultrastable peptides are being pursued as pharmaceutical tools and drug leads, particularly as scaffolds onto which bioactive epitopes can be grafted [1, 2]. Cyclotides are also being employed in the agricultural industry as natural pesticides, with Sero-X, a cyclotide-based pesticide, recently receiving regulatory approval and becoming commercially available in Australia [3]. Thus, in general, there is a requirement for the efficient, cost-effective synthesis of cyclotides and other disulfide-rich macrocyclic peptides compatible with both synthetic and recombinant expression routes. Chemical approaches to the head-to-tail cyclization of disulfide-rich peptides have proven valuable for a wide range of research studies but are, nevertheless, limited by their requirement for toxic, corrosive, or specialized reagents that are associated with

Timo Nuijens and Marcel Schmidt (eds.), Enzyme-Mediated Ligation Methods, Methods in Molecular Biology, vol. 2012, https://doi.org/10.1007/978-1-4939-9546-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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low-yielding and/or expensive production processes [4–6]. The use of sortase A for the chemoenzymatic ligation of disulfide-rich peptides offers an affordable and efficient approach to peptide cyclization. Sortase A (SrtA) is a bacterial enzyme, originally isolated from Staphylococcus aureus, that is now applied as a biochemical tool. It contains a cysteine in the active site, recognizes substrates containing an LPXTG motif (where X can be any proteogenic amino acid) and hydrolytically cleaves between the threonine and glycine, forming a threonyl-SrtA intermediate that is resolved in the presence of a polyglycine nucleophile to ligate the substrate to the polyglycine containing moiety (Fig. 1a). The ability of SrtA to form amide bonds has been utilized for a wide range of protein and peptide engineering applications, including N- and C-terminal labeling [7–10], peptide PEGylation [11], and the production of macrocycles, where peptides are strategically designed to contain the polyglycine nucleophile at the N-terminus, and the LPXTG “sortagging” motif at the C-terminus [12, 13]. The protocol we present here describes the use of sortase A in the cyclization of disulfide rich peptides, using an optimized SrtA pentamutant (P94R/D160N/D165A/K190E/K196T) (abbreviated henceforth as SrtA5
), which has a reported increase in enzymatic efficiency of approximately 120-fold compared to wild type (wt) SrtA [14]. As described in the protocol, wt SrtA may be used if slower reactions are deemed to be optimal for specific cyclization reactions. For illustration, we use the prototypic cyclotide kalata B1 as an example of a disulfide-rich cyclic peptide, but the protocol is also applicable to the intermolecular ligation of noncyclic disulfide-rich peptides [15]. The protocol involves an approach where the disulfide-rich peptides are oxidized prior to cyclization to facilitate obtaining the final product with minimal amounts of misfolded isomers; however, the option to cyclize the peptides first, followed by the formation of the disulfide bridges is also possible, and can be achieved by adding a reducing agent like tris(2-carboxyehyl)phosphine hydrochloride (TCEP) or 1,4-dithiothreitol (DTT) to maintain the peptides in a reduced state during cyclization [13].

2

Materials

2.1 Fmoc SolidPhase Peptide Synthesis

Perform synthesis in a glass column containing a stopcock and a fritted filter attached to a vacuumed flask for waste collection. Carefully and responsibly dispose of all waste according to sitespecific regulations. 1. Glass column containing a stopcock and fritted glass filter for solid phase peptide synthesis.

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31

Fig. 1 Schematic for sortase A-mediated cyclization of disulfide-rich peptides. (a) Cartoon showing a linear disulfide-rich peptide (sequence of kalata B1) with an N-terminal polyglycine and the C-terminal containing the native residues LPV and additional TGG to form the LPXTGG sortase recognition motif. The process of sortase A cyclization is also shown including recognition of the sortagging sequence and cleavage between T/G to form an intermediate that is resolved by the N-terminal polyglycine to form the final cyclized. (b) Representation of the cyclization trials that facilitate tailoring the sortase A ligation to specific peptides by varying ratio of peptide–sortase and temperature. Samples at fixed time-points should be collected and monitored using RP-HPLC and MS. (c) Once peptide-specific concentrations and temperatures have been determined scale up the cyclization reaction, remove His6-tagged sortase A by centrifuging with Ni2+-NTA, filter, purify, and lyophilize the final product

2. 2-chlorotitryl resin (0.8 mmol/g substitution value, 0.1 mmol scale). 3. Fmoc-protected amino acids.

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4. Activating agent: 0.5 M O-(1H-6-chlorobenzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) in dimethylformamide (DMF). 5. Base: N,N,-diisopropylethylamine (DIPEA). 6. Capping solution: methanol (MeOH)–DIPEA–DCM (2:1:17, v/v/v) for first coupling reaction. 7. Resin wash solvents: DMF and dichloromethane (DCM). 8. Fmoc deprotection solution: piperidine in DMF (1:2, v/v). 9. Cleavage solution: 95% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) water, 2.5% (v/v) triisopropylsilane. 10. Peptide extraction solutions: diethyl ether and 45% (v/v) acetonitrile (ACN), 0.01% (v/v) TFA in water. 11. Dry ice to flash-freeze samples prior to lyophilization. 2.2 Peptide Oxidative Folding

1. Solution to dissolve the peptide prior to oxidation: 50% (v/v) isopropanol in water. 2. Oxidation buffer for kalata B1: 50% (v/v) isopropanol, 0.1 M ammonium bicarbonate pH 8.0 and 1 mM reduced glutathione in water. 3. Reaction quenching solution: 1% (v/v) TFA in water.

2.3 Peptide Purification

Dissolve peptides in high performance liquid chromatography (HPLC) solvents A and/or B (see below) and filter using 0.45 μm filter before loading onto column for purification. 1. Reverse phase (RP)-HPLC C18 columns for proteins and peptides. 2. HPLC Solvent A: 0.05% (v/v) TFA in ultrapure water, filter through a 0.45 μm filter and store at room temperature. 3. HPLC Solvent B: 0.05% (v/v) TFA and 90% (v/v) acetonitrile (ACN) in ultrapure water, filter through a 0.45 μm filter and store at room temperature. 4. HPLC Solvent A/B: 0.05% (v/v) TFA and 45% (v/v) ACN in ultrapure water, filter through a 0.45 μm filter and store at room temperature. 5. LC/MS Solvent A: 0.05% (v/v) formic acid in ultrapure water, filter through a 0.45 μm filter and store at room temperature. 6. LC/MS Solvent B: 0.05% (v/v) formic acid and 90% (v/v) ACN in ultrapure water, filter through a 0.45 μm filter and store at room temperature.

2.4 Sortase A Expression and Ligation Material

SrtA and SrtA5
are commercially available (see Note 1) or can be produced by recombinant expression. Removal of the 59-amino acid transmembrane domain from the N-terminal of SrtA (SrtA

Sortase A for the Synthesis of Disulfide-Rich Macrocycles

33

Δ59) leaves the soluble catalytic domain of SrtA which is ideal for the transpeptidation reaction [16]. Additional materials are listed below: 1. SrtA5
expression plasmid (pET29) or SrtA Δ59 expression plasmid SrtA (pET28a+) (see Note 1). 2. E. coli BL21(DE3) cells. 3. Luria–Bertani (LB) medium agar plates: 10 g/L peptone, 5 g/ L yeast extract, 5 g/L sodium chloride, 12 g/L agar, and 50 μg/mL kanamycin. 4. LB broth medium: 20 g/L peptone, 5 g/L yeast extract, 5 g/L sodium chloride, and 50 μg/mL kanamycin. 5. Isopropyl-β-D-1thiogalactopyranoside (IPTG) 1 M stock solution in water (sterile filtered). 6. Lysis buffer: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, and 20 g/L DNase I. 7. Elution buffer: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 500 mM imidazole, 10% (v/v) glycerol. 8. Sortagging buffer stock solution: 0.5 M Tris–HCl, 1.5 M NaCl, 0.1 M CaCl2. Adjust to pH 8 using 0.5 M HCl or 0.5 M NaOH. Store at 4
C for up to 3 months (see Note 2). 9. Working sortagging buffer: 0.05 M Tris–HCl, 0.15 M NaCl, 0.01 M CaCl2. Adjust to pH 8 using 0.5 M HCl or 0.5 M NaOH. Make fresh on day of experiment. 10. Sortagging quenching solution: 1% (v/v) TFA in water. 11. Nickel-nitriloacetic acid (Ni2+-NTA) agarose sludge is available commercially. To prepare the agarose sludge prior to separation of His6-tagged SrtA, see Subheading 3.6. 2.5 Additional Equipment

1. Round bottom flasks. 2. Magnetic stirrer and stirrer bars. 3. Vacuum rotary evaporator. 4. Lyophilizer. 5. NanoDrop UV-Vis spectrophotometer. 6. Eppendorf tubes (1.5 and 2 mL capacity). 7. Centrifuges: table top microcentrifuge and large capacity centrifuge. 8. Reverse phase high performance liquid chromatography (RP-HPLC) instrument. 9. LC-electrospray-mass spectroscopy (ESI-MS) instrument. 10. Incubator with shaker. 11. Cell disruptor.

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12. UV-Vis spectrophotometer. 13. 5 mL HisTrap™ Ni2+-NTA FF column (available from GE Healthcare). 14. 10 kDa centrifugal ultrafiltration unit.

3

Methods

3.1 Peptide Assembly Using Fmoc Solid-Phase Peptide Synthesis

Synthesize the desired peptide to contain an N-terminal polyglycine (at least two glycine residues) and the LPXTGG sortase A recognition motif at the C-terminus (see Note 3 and Fig. 1a for sequence of kalata B1). 1. Add 0.125 g of 2-chlorotitryl resin (0.8 mmol/g substitution value, 0.1 mmol scale) to a glass column containing a stopcock and a fritted glass filter (see Note 4). Soak in 5 mL DCM for 20 min, then drain the DCM by vacuum filtration. 2. Prepare the first amino acid (Fmoc-Gly-OH) for coupling by completely dissolving the amino acid (2 equiv.) in 5 mL DCM and DIPEA (8 equiv.). Add the coupling solution to the resin and allow the coupling reaction to proceed for 1 h. 3. Open the stopcock on the column filter and drain coupling solution into a waste flask under vacuum, wash with 5 mL DCM–MeOH–DIPEA resin capping solution three times (see Note 5), then with 5 mL DCM three times and finally with 5 mL DMF three times (see Note 6). 4. Remove the Fmoc protecting group using 5 mL DMF/piperidine deprotecting solution for 5 min two times. Wash with 5 mL DCM three times and finally with 5 mL DMF three times. 5. Prepare subsequent amino acids for coupling using amino acid (4 equiv.), HCTU (4 equiv.) and DIPEA (8 equiv.) in 5 mL DMF, add the coupling solution to the resin and couple for 10 min. Repeat the coupling step. Wash with 5 mL DCM three times, followed by 5 mL DMF three times. 6. Repeat steps 4 and 5 until all amino acids are coupled. 7. Remove final Fmoc group as in step 4, wash resin (one time 5 mL DCM, then one time 5 mL DMF) three times and finally with 5 mL DCM three times. Subsequently, dry the resin completely under a flow of N2. 8. Release peptide from resin and simultaneously cleave amino acid side chain protecting groups by incubating for 2 h in the TFA/triisopropylsilane/water peptide cleavage solution using a volume of 20 mL/g dry resin. Concentrate the peptide using a vacuum rotary evaporator.

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35

9. Add ice-cold diethyl ether (50 mL/g dry resin) to precipitate the peptide and separate the cleavage material (in solution) from the desired peptide using a column filter. 10. Dissolve the peptide in solvent A/B, collect the solution in a round bottom flask via filtration using a fritted column filter and remove the excess of organic solvent on a vacuum rotary evaporator. Flash-freeze the solution using dry ice and lyophilize the peptide. Confirm the correct product using ESI-LC/ MS (see Note 7), purify crude peptide using RP-HPLC, lyophilize and store at 20
C prior to oxidation. 3.2 Peptide Oxidative Folding

Oxidation is peptide specific (see Note 8). The following is a description of the oxidation of kalata B1. 1. Dissolve the purified peptide in 50% (v/v) isopropanol–water to a tenth of the final oxidation buffer volume (see Note 9), and leave stirring at 200 rpm using a stir bar and a magnetic stirrer while preparing the oxidation buffer. 2. Prepare the oxidation buffer (see Note 9), then add the peptide dropwise to the oxidation buffer. Leave stirring and monitor reaction at fixed time points (e.g., T1 h, T8 h, T16 h, T24 h) by diluting 10 μL samples in 10 μL solvent A and analysis via LC/MS (0–70% solvent B gradient) (see Note 10). 3. Quench the complete reaction mixture by acidification to pH 4 using a solution of 1% (v/v) TFA in water, dilute to less than 10% (v/v) isopropanol using solvent A, filter (0.45 μm) and purify via RP-HPLC. Analyze fractions using ESI-MS to obtain folded peptide (see Note 10), pool relevant fractions, flashfreeze using dry ice, lyophilize, and store at 20
C prior to ligation.

3.3 Expression of SrtA and SrtA5


These methods have been adapted from refs. 9, 17, 18. 1. Put 50 μL of competent E. coli BL21(DE3) cells into a 1.5 mL Eppendorf tube, keep on ice and add 100 ng SrtA or SrtA5
plasmid (pET28a+ or pET29a, respectively), tap gently and incubate on ice for 10–15 min. 2. Incubate the cells at 42
C for 30 s, then put on ice for 1 min before adding 500 μL LB medium prewarmed to 42
C. Incubate for 1 h at 37
C while shaking at 200 rpm. 3. Centrifuge the cell containing medium at 7000  g, discard supernatant and resuspend in 20 μL LB medium. 4. Streak the cells onto an LB medium agar plate and incubate upside down overnight at 37
C. 5. Inoculate one colony in 100 mL LB medium and incubate overnight at 25
C while shaking at 200 rpm.

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Akello J. Agwa et al.

6. Dilute culture to 1 L and incubate overnight in LB medium at 25
C while shaking at 200 rpm. Regularly measure the OD600 using a UV/Vis spectrophotometer to obtain an OD600 ~ 0.7. 7. At OD600 ~ 0.7 induce protein expression by adding 0.5 mL of 1 M IPTG (to 1 L of medium) to make a final concentration of 0.5 mM IPTG and incubate for 3 h at 37
C. 8. Centrifuge at 7000  g at 4
C for 10 min to harvest cells. 9. Resuspend cell pellet in ice cold lysis buffer and lyse by passing through a cell disrupter at 26,000 psi. 10. Separate protein from cell debris by centrifuging at 12,000  g at 4
C for 30 min, collect supernatant and keep on ice prior to purification. 11. To purify the protein, wash the 5 mL HisTrap™ Ni2+-NTA FF column with lysis buffer (ten times 5 mL washes; gravity flow). Load the protein (supernatant from step 10) to the column using gravity flow and wash with lysis buffer (20 washes with 5 mL; gravity flow). 12. Elute the protein using elution buffer (five times 5 mL; gravity flow), collect fractions containing SrtA (or SrtA5
) and concentrate the protein using the 10 kDa centrifugal filter at 14,000  g for 30 min, to a tenth of the volume. 13. Aliquot into 500 μL portions and quantify using UV/Vis spectrophotometry (see Note 11), flash-freeze, and store at 80
C. 3.4 Tailoring the Ratio of Linear Peptide–SrtA5
and Temperature to Specific Disulfide-Rich Peptides

1. Prepare working buffer in ultrapure water and vary peptide–SrtA5
in molar ratios of 1:1, 2:1, and 3:1 using 10–150 μM peptide and SrtA5
(see Notes 11–14) (Fig. 1b). Confirm that pH ¼ 8 and adjust if necessary using 0.5 M HCl or 0.5 M NaOH to final trial reaction volumes of 250 μL in 1.5 mL Eppendorf tubes (see Note 15). Perform reactions at 25
C and use a magnetic stirrer and stirrer bar to mix at 200 rpm. 2. Collect 10 μL samples from reaction at T0 min, T5 min, T15 min, T30 min, T1 h, T2 h, and T24 h and add 10 μL of 1% (v/v) TFA to quench the reaction. 3. Load time-point samples onto a RP-HPLC C18 column, monitor at 215 nm and/or at 280 nm (for peptides containing aromatic residues). Collect eluent from each peak and analyze masses from each peak using ESI-MS to identify products (see Notes 16 and 17). 4. Once ideal ratio of peptide–SrtA5
has been achieved (see Note 18), optimize reaction speed by varying temperature while keeping SrtA5
and buffer concentration and ratio of peptide–SrtA5
constant. Monitor reactions at 4, 25 and

Sortase A for the Synthesis of Disulfide-Rich Macrocycles

37

37
C with and without stirring (see Note 19). Collect and prepare time point samples (as done for step 2 above) (see Note 19), Monitor and analyze the samples using analytical RP-HPLC and ESI-MS (as done for step 3 above). 3.5 Scale-Up of the Enzymatic Cyclization Reaction

Final volumes for larger scale reactions will be dependent on the desired final amount of peptide (see Notes 20 and 21) (Fig. 1c). 1. Prepare working sortagging buffer in ultrapure water and use optimized specific ratio of peptide–SrtA5
, optimal temperature and duration as determined during prior trials (see Subheading 3.1 above). 2. At the predetermined optimal end-point (see Subheading 3.1 above) slow down the reaction by placing reaction vessel on ice and lowering the pH to 90–95%). Omniligase-1 recognizes four amino acids at the C-terminus of an acyl donor fragment (position P4–P1; according to Schechter and Berger) [13] and two N-terminal amino acids, that is, P10 and P20 , of a nucleophilic acyl acceptor fragment (amine nucleophile). Due to its broad substrate scope omniligase-1 tolerates a wide range of different amino acids sequences (see Subheading 4), hence resulting in a footprint-free coupling reaction. Since no fixed enzyme recognition sequence is required, the ligation can be considered to be intrinsically traceless. In conclusion, omniligase-1 mediated ligation represents a powerful tool for inter- and intra-molecular ligation of various peptides, both at lab scale as well as at larger scale for commercial manufacture. In this chapter we describe the use of omniligase-1 for the scalable assembly of the therapeutic glucagon-like peptide-1 (GLP-1) analog exenatide and the one-pot head–tail cyclization and oxidative folding of naturally occuring disulfide-rich macrocylic peptide, the cyclotide MCoTI-II. These two key examples display representative guidelines for the assembly of a wide variety of different linear and cyclic peptides using omniligase-1.

Omniligase-1-Mediated Peptide Ligation and Cyclization

2

45

Materials Prepare all solutions using ultrapure water (ddH2O; prepared by purifying deionized water, to attain a sensitivity of 18 MΩ cm at 25  C). All reagents and stock solutions should be stored at the indicated temperatures. Diligently follow all waste disposal regulations when disposing of waste materials. Wear appropriate protective clothing and safety glasses. Work with volatile compounds should be performed in a fume hood.

2.1 Intermolecular Peptide Ligation: CEPS of Exenatide

1. Ligation buffer: 1 M potassium phosphate, pH 8.5. Weigh out 16.38 g dipotassium phosphate (K2HPO4) and 0.82 g monopotassium phosphate (KH2PO4) in a 100 mL graduated glass cylinder. Add 80 mL of ddH2O and dissolve the corresponding phosphate salts. Gently stir the buffer solution and adjust the pH with potassium hydroxide (KOH, 1 M) solution or phosphoric acid (H3PO4, 1 M) to pH 8.5. Finally, fill to a total volume of 100 mL. The buffer can be stored at 25  C for 6 months. A buffer solution of 1 M Tricine pH 8.5 can be used as an alternative (see Note 1). 2. Reducing reagent (irreversible) stock solution: 350 mM tris (2-carboxyethyl)phosphine (TCEP), pH 8.2–8.4. Weigh out 100 mg TCEP and dissolve in 800 μL ddH2O. Mix and adjust pH to 8.2–8.4 using a 1 M KOH solution (see Note 2). Fill to a total volume of 1 mL and aliquot into 50 μL portions. Store at 20  C for 6 months. Avoid excessive freeze and thaw cycles. 3. Reducing agent (reversible) stock solution: 100 mg/mL dithiothreitol (DTT), pH 8.2–8.4. Weigh out 100 mg DTT and dissolve in 800 μL ddH2O. Mix and adjust pH to pH 8.2–8.4. Fill to 1 mL of total volume and aliquot into 50 μL portions. The buffer can be stored closed from air at 20  C and avoid excessive freeze and thaw cycles. 4. Enzyme: Omniligase-1 [1, 10, 12]. Available from EnzyPep B.V. 5. Synthesized peptides to be ligated: H-Exenatide(1–21)OCam-L-OH (Peptide 1) and H-Exenatide(22–39)-NH2 (Peptide 2) (Table 1). Peptides can be prepared according to standard literature procedures [4] and C-terminal (Cam-)ester peptides can be prepared according to Nuijens et al. [14]. Alternatively, peptides can be purchased from various commercial sources. 6. HPLC-MS system for peptide analysis (e.g., Agilent 1260 Infinity series HPLC with Agilent 6130 Quadrupole mass spectrometer or equivalent).

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Table 1 Sequence and molecular weight (MW) of exenatide fragments H-Exn (1–21)-OCam-Leu-OH and H-Exn(22–39)-NH2

Name Sequence

H-Exenatide(1–21)-OCam-Leu-OH a

H-HGEGTFTSDLSKQMEEEAVRL-OCam-L-OH

MW (g/mol)

2535.8

Name

H-Exenatide(22–39)-NH2

Sequence

a

MW (g/mol)

H-FIEWLKNGGPSSGAPPPS-NH2 1840.1

a

Amino acids recognized by omniligase-1 in bold

7. Analytical C18 HPLC-column (e.g., Phenomenex C18, 5 μm particle size, 250  4.6 mm or equivalent). 8. Mobile phase A (analytical): ddH2O + 0.05% methanesulfonic acid (MSA) (v/v). 9. Mobile phase B (analytical): acetonitrile + 0.05% (v/v) MSA. 10. Preparative C18 HPL column (e.g., Phenomenex Luna 10 μM Prep C18, 250  50 mm or equivalent). 11. Mobile phase A (preparative): ddH2O + 0.05% trifluoroacetic acid (TFA) (v/v). 12. Mobile phase B (preparative): acetonitrile + 0.05% (v/v) TFA. 13. pH meter (calibrated) with microelectrode for determining the pH of the reaction mixture. 14. Reaction vials (e.g., glass vials (1–50 mL)) or microcentrifuge tubes. 15. Ligation quenching (48:48:4, v/v/v).

solution:

ddH2O–acetonitrile–MSA

16. Potassium hydroxide (KOH) solution: 1 M in ddH2O.

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Table 2 Sequence and molecular weight (MW) of the linear ester H-MCoTI-IIOCam-Leu-OH

Name Sequence

H-MCoTI-II-OCam-Leu-OH a

H-ILKKCRRDSDCPGACICRGNGYCGSGSDGGVCPKOCam-Leu-OH

MW 3648.2 (g/mol) a

Amino acids recognized by omniligase-1 in bold

2.2 Peptide Head-toTail Cyclization: CEPS of MCoTI-II

3

Identical materials needed as compared to Subheading 2.1, except the following: 1. Linear substrate: H-MCoTI-II-OCam-Leu-OH (Table 2). MCoTI-II-OCam-L-OH contains both the acyl acceptor and acyl donor sequence in one fragment, that is, it can be denoted as P10 -P20 -Peptide-P4-P3-P2-P1-OCam-Leu-OH. It can be prepared according to Schmidt et al. [10] or purchased from commercial sources.

Methods For omniligase-1 mediated ligation there are two possibilities to perform a ligation: (a) Intermolecular ligation between two peptide fragments (see Subheading 3.1). (b) Intramolecular ligation (head-to-tail cyclization) (see Subheading 3.2). The following protocols describe a laboratory scale reaction, which can, depending on the experimental needs, be scaled using adapted protocols. Intermolecular ligation has been proven at >50 g and intramolecular ligation on a multigram scale.

3.1 Intermolecular Peptide Ligation: CPES of Exenatide

In this example of an omniligase-1-catalyzed intermolecular peptide ligation, H-Exenatide(1–21)-OCam-L-OH and H-Exenatide

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Fig. 2 Schematic illustration of omniligase-1 catalyzed ligation of H-Exn(1–21)-OCam-L-OH with H-Exn (22–39)-NH2

(22–39)-NH2 are used as representative acyl donor and acyl acceptor fragments, respectively (Fig. 2). The general sequence of the acyl donor fragments (e.g., H-Exn (1–21)-OCam-L-OH) can be defined as Peptide-P4-P3-P2-P1OCam-Y-OH, with Y generally being Leu (see Note 3). Only the four C-terminal amino acids P4–P1 are recognized by omniligase-1. Omniligase-1 also recognizes the first two N-terminal amino acids of the acyl acceptor fragment and thus the general sequence for the acyl acceptor fragment (e.g., H-Exn(22–39)-NH2) can be defined as P10 -P20 -peptide. In the case of exenatide, omniligase-1 recognizes the N-terminal amino acids FI (P10 , P20 ) of H-Exn(22–39)-NH2 and P4–P1 are corresponding to amino acids AVRL of H-Exn (1–21)-OCam-L-OH. Since the N-terminal sequence of fragment H-Exn(1–21)-OCam-L-OH (i.e., HG) is only poorly recognized by omniligase-1 no N-terminal protection is being required to avoid cyclization or multimerization of the acyl donor fragment (see Fig. 7 and Note 4). The general substrate preferences of omniligase-1 in the respective pockets (P4–P1, P10 –P20 ) are shown in Figs. 6 and 7. There are no specific requirements beyond residues outside this six amino acid recognition sequence (P4 through to P20 ), D-amino acids as well nonpeptidic moieties (e.g., polyethylene glycol (PEG)) can be included in the peptide outside the enzyme recognition motifs. In addition, several nonnatural amino acids are tolerated by omniligase-1 within the recognition sequence of Omniligase-1. Apart from the ligation of exenatide fragments H-Exn(1–21)-

Omniligase-1-Mediated Peptide Ligation and Cyclization

49

Fig. 3 HPLC-chromatograms of the ligation of H-Exn(1–21)-OCam-L-OH to H-Exn(22–39)-NH2 (1.2 equiv.). Results are shown after the ligation has proceeded for 10, 30, 60, and 90 min. After 90 min full consumption of H-Exn (1–21)-OCam-L-OH and formation of H-Exn(1–39)NH2 (96%) is observed. Four percent of the hydrolysis side product H-Exn(1–21)-OH is formed

OCam-L-OH and H-Exn(22–39)-NH2, omniligase-1 enables the efficient ligation of many different peptide fragments with various sequences. For peptide fragemts that differ from the representative model peptides (i.e., H-Exn(1–21)-OCam-L-OH and H-Exn (22–39)-NH2) the optimal ligation position/sequence should be chosen based data given in Figs. 6 and 7 (see Notes 5, 6 and 7). H-Exn(1–21)-OCam-L-OH and H-Exn(22–39)-NH2 have m/z values of 845.72+/634.53+/507.84+ and 920.52+/614.03+/ 460.74+, respectively. A mass of 4186.6 is observed for the ligated product H-Exenatide(1–39)-NH2 (H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2), which elutes at 13.3 min in the RP-HPLC system (Fig. 3). The intermolecular ligation reaction is typically completed within 90 min with a ~96% yield (HPLC-based) after complete consumption of the acyl donor fragment H-Exn(1–21)-OCam-L-OH. As a side product, hydrolysis of H-Exn(1–21)-OCam-L-OH to the corresponding H-Exn (1–21)-OH free acid (4%) occurs. The protocol for a ligation of purified fragments (>95% HPLC purity) is described below. 1. Weigh out 10.5 mg (3.5 μmol) of purified, lyophilized H-Exn (1–21)-OCam-L-OH (M(.4TFA) ¼ 2991.9 g/mol) and 8 mg

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Marcel Schmidt and Timo Nuijens

H-Exn(22–39)-NH2 (1.1 equiv., M(.2TFA) ¼ 2068.1 g/mol) and place in a 2 mL glass vial (see Notes 8 and 15). 2. Thaw omniligase-1 aliquot and reducing agent stock solution (TCEP or DTT). 3. Dissolve the fragments in 335 μL of ligation buffer and 40 μL acetonitrile to a final concentration [H-Exn(1–21)-OCam-LOH] of 9.3 mM (see Note 9). Make sure the fragments are properly dissolved (see Note 11). In case of low fragment solubility, please see Note 10. 4. Upon dissolving of the fragments, rapidly proceed with steps 5–8 (see Note 12). Please make sure that there are no noncompatible reagents present in the reaction mixture (see Note 13). 5. Add 18.75 μL of the TCEP stock solution to a final concentration of 5 mg/mL (17.5 mM). Alternatively DTT can be used instead of TCEP at the same concentration (see Note 16). 6. Measure the pH of the reaction mixture. The pH should be between 8.0 and 8.3. If the pH is below 8.0, adjust by adding suitable amounts of 1 M KOH solution (see Notes 13 and 14). 7. Take a blank reference sample for HPLC-MS analysis: add 5 μL of the peptide reaction mixture to 95 μL of quenching solution (see Notes 17 and 18). Analyze the sample using HPLC-MS with a linear gradient of 25–65% B in 21 min, a column temperature of 40  C and a flow rate of 1 mL/min. 8. Add 80 μg of omniligase-1 (0.0008 molar equivalents as compared to ester fragment)—see Note 20. Aliquot omniligase-1 upon first use and avoid excessive freeze–thaw cycles (see Note 19). 9. Incubate the reaction mixture at room temperature (20–25  C) for 90 min (see Note 21). 10. Quench a sample of the enzymatic ligation mixture after 10, 30, 60, and 90 min: add 5 μL of the sample to 95 μL of quenching solution. 11. Monitor the progress of the reaction by HPLC-MS using the gradient method as in step 7. Full consumption of the ester fragment (H-Exn(1–21)-OCam-L-OH) and almost quantitative product formation should be observed within 90 min (see Note 22). The product (H-Exn(1–39)-NH2) mass of 4186.6 Da (m/z 1047.74+/838.35+/698.86+/599.17+) should be observed. The hydrolyzed ester fragment (H-Exn (1–21)-OH) appears as a side product with a mass difference of 171.2 Da as compared to the Cam-ester starting material (H-Exn(1–21)-OCam-L-OH) (Fig. 3). 12. After the reaction is completed, stop the enzymatic reaction by addition of TFA to a final pH between 1 and 3.

Omniligase-1-Mediated Peptide Ligation and Cyclization

51

13. Filter the solution of crude peptide with a hydrophobic membrane filter before injection into the HPLC for final analysis or purification to remove potentially insoluble impurities. 14. Purify the ligation product (H-Exn(1–39)-NH2) via preparative reversed-phase (RP)-HPLC. Use a gradient of 25–45% mobile phase B (preparative) in 21 min, a flow rate of 45 mL/min with a column temperature of 20  C. 15. Pool pure product containing fractions, flash-freeze in liquid nitrogen and lyophilize the samples for further characterization. 3.2 Peptide Head-toTail Cyclization: CEPS of MCoTI-II

In this particular example, the head-to-tail cyclization of the disulfide-rich peptide MCoTI-II (H-ILKKCRRDSDCPGACICR GNGYCGSGSDGGVCPK-OCam-Leu-OH, M ¼ 3648.2 g/mol) is described. The intramolecular ligation using omniligase-1 is followed by oxidative folding into the native state of MCoTI-II using a one-pot procedure (see Fig. 4) [10]. The linear peptide Cam-ester corresponds to the general overall structure H-P10 -P20 -peptide-P4-P3-P2-P1-OCam-Y-OH, with Y generally being Leu (see Note 3). In case of MCoTI-II this corresponds to H-ILpeptide-VCPK-OCam-L-OH. For peptide sequences other than the sequence given above please refer to Subheading 3.1 for choosing the optimal ligation site for your peptide. Open chain (oc, linear reduced) MCoTI-II has a molecular weight of 3648.2 g/mol. A mass of 3456.5 g/mol should be observed for the cyclic reduced cMCoTI-II and 3450.5 g/mol for the cyclic oxidized (folded) form cf-MCoTI-II, which elutes at tR ¼ 9.8 min in the RP-HPLC (Fig. 5). The head-to-tail cyclization reaction should be completed within 30 min with an >90% yield (HPLC-based conversion to product). As a side product, hydrolysis of the linear H-MCoTI-II-OCam-Leu-OH to the corresponding acid is observed (H-MCoTI-II-OH; 5%). 1. Weigh out 1.1 mg of purified, lyophilized oc-MCoTI-II Cam-ester (M(.7TFA) ¼ 4446.7 g/mol, >90% HPLC purity) in a 2 mL glass vial (see Note 8). 2. Thaw an aliquot of omniligase-1. 3. Dissolve the fragments in 1000 μL ligation buffer to a final substrate concentration [oc-MCoTI-II-OCam-Leu-OH] of 0.25 mM (see Note 23). 4. Upon dissolving the fragment, rapidly proceed with steps 5–8 (see Note 12). Please make sure there are no noncompatible reagents present in the reaction mixture (see Note 13). 5. Monitor the pH of the reaction mixture. The pH should be between 8.0 and 8.3. If the final pH is below 8.0, adjust the pH

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Fig. 4 Reaction scheme for the cyclization and oxidative folding of open chain (oc)-MCoTI-II in a one-pot reaction to yield cyclic, native folded (cf)-MCoTI-II

Fig. 5 One-pot peptide head-to-tail cyclization of (oc)-MCoTI-II to cyclic folded (cf)-MCoTI-II. HPLC chromatograms (λ ¼ 220 nm) after 0 min (oc-MCoTI-II), 30 min (cyclic (c)-MCoTI-II), and 14 h (cf)-MCoTI-II. The correct folding of cf-MCoTI-II was confirmed via NMR structure determination

by adding suitable amounts of 1 M NaOH solution (see Notes 13 and 14). 6. Take a blank reference sample for HPLC-MS analysis: add 10 μL of the peptide reaction mixture to 90 μL of quenching solution (see Notes 17 and 18). Analyze the sample with a linear gradient of 5–40% mobile phase B (analytical) in 21 min at a flow rate of 1 mL/min and a column temperature of 40  C.

Omniligase-1-Mediated Peptide Ligation and Cyclization

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7. Add 10 μg of omniligase-1 (0.001 molar equivalents as compared to linear ester fragment). Aliquot omniligase-1 upon first use and avoid excessive freeze–thaw cycles (see Note 19). 8. Incubate the reaction mixture at room temperature (20–25  C) for 30 min. 9. Quench a sample of the enzymatic ligation mixture (after 10, 20, and 30 min): add 10 μL of the sample to 90 μL of quenching solution. Monitor the progress of the reaction by HPLC-MS. A mass shift of 189 Da should be observed upon conversion of a target peptide from the linear (oc-MCoTI-II; m/z 912.44+, 730.15+, 608.66+) to its reduced circular form (c-MCoTI-II; m/z 865.1, 692.3, 577.1). The reaction should be completed with formation of (c)-MCoTI-II after 30 min in over >90% HPLC conversion to product. In order to obtain the cyclic reduced product, the reaction mixture can be purified via preparative HPLC at this stage. In case of the latter, purify the cyclic reduced product using a gradient from 5% to 40% mobile phase B (preparative) in 30 min at a flow rate of 45 mL/min and a column temperature of 20  C. 10. For oxidative folding add reduced glutathione (GSH; 1.5 mg) to a final concentration of 5 mM to the reaction mixture. The presence of reduced GSH facilitates disulfide bond formation and the formation of the desired product, (cf)-MCoTI-II. Ensure that the pH of the reaction mixture remains between 8 and 8.5 upon addition of reduced GSH and adjust the pH accordingly using 1 M KOH. 11. Add a magnetic stirring bar to the reaction mixture and gently stir overnight (12–18 h) exposed to air. 12. After overnight incubation, quench a sample of the enzymatic ligation mixture for HPLC-MS analysis: add 10 μL of the sample to 90 μL of quenching solution. Alternatively, the reaction mixture can be sampled and injected into the HPLC-MS directly. 13. Monitor the progress of the reaction by HPLC-MS. The formation of cyclic folded cf-MCotI-II is observed by a mass difference of 6 Da and a retention shift (from tR ¼ 14.5 min to tR ¼ 9.8 min, Fig. 5) in the HPLC spectrum. If the oxidative folding is not yet completed after 18 h, see Note 24. 14. After the reaction has completed, stop the reaction by addition of acetonitrile to a percentage of 5% (v/v) and TFA to a concentration of 0.05% (v/v). 15. Filter the solution of crude peptide with a hydrophobic membrane filter before injection into the HPLC for analysis or purification to remove potentially insoluble impurities.

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16. Purify the ligation product ((cf)-MCoTI-II) via preparative reverse-phase (RP)-HPLC using a gradient from 5% to 40% mobile Phase B (preparative) in 30 min at a flow rate of 45 mL/min and a column temperature of 20  C. 17. Pool pure product containing fractions, flash-freeze in liquid nitrogen and lyophilize the samples for further characterization. The one-pot cyclization and folding reaction can be scaled to grams (see Note 25). When translating this protocol to other disulfide-rich peptides, please be aware that the one-pot cyclization and oxidative folding does not work for all disulfide-rich peptides. In many cases, orthogonal protection/deprotection is required in order to obtain the correct disulfide bond pattern. In addition, for certain disulfide-rich peptides the optimal conditions for oxidative folding differ from the enzymatic cyclization conditions (e.g., buffer type, addition of cosolvents) and need to be adapted accordingly. Alternatively, after omniligase-1 cyclization, TCEP or DTT is added followed by preparative HPLC purification of the cyclic, reduced product. After lyophilization, optimized folding conditions can be applied in order to obtain the final product.

4

Notes 1. In case of poor solubility when using different peptide sequences: besides the commonly used potassium phosphate buffer tricine buffer can also be used. Using a tricine-based buffer system, substrates often show an increased solubility as compared to phosphate buffered solutions. However, the ligation results might slightly differ from using a phosphate buffered system and, when used in high concentrations, tricine can lead to side reactions (e.g., transesterification). Another option (in the case of hydrophobic peptides) is to lower the buffer strength (e.g., to 100 mM or lower). Additional alternatives are described in Note 10. 2. If the pH of the TCEP stock solution is not adjusted to pH 8.0 prior to the reaction, the addition of TCEP stock solution to the ligation mixture can significantly reduce the pH and negatively influence the enzymatic ligation. 3. As a standard procedure, it is recommended to use an elongated glycolate-Leu-OH ester. In particular cases the leucine can be replaced by other amino acids, which, however, needs to be evaluated individually for each case. If a nonelongated ester is desired, a carboxamidomethyl (Cam)-ester (O-CH2CONH2) can be used. The corresponding glycolic acid

Omniligase-1-Mediated Peptide Ligation and Cyclization

55

(O-CH2-COOH) ester without any elongation bearing a free acid at the C-terminus is not suitable. 4. If amino acids are present in acyl donor fragment at the N-terminus in positions P10 and P20 that are not or very poorly recognized by omniligase-1, no N-terminal protection is necessary. If this is not the case and well recognized amino acids are present, N-terminal protection of the acyl donor (ester) fragment is mandatory. For example, as a permanent, noncleavable N-terminal protecting group an acetyl-group is suitable. As an alternative, orthogonal cleavable groups such as phenylacetyl (PhAc) can be used. PhAc can be removed with commercially available acylases (e.g., immobilized PenG acylase). In addition, several other N-terminal chemically cleavable protection groups can be used (Dde (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)), carboxybenzyl (Cbz), trifuloroacetyl (TFA), tert-butyloxycarbonyl (Boc), etc.). Alternatively, more selective peptiligase variants other than omniligase-1 could be used (e.g., thymoligase) [15]. These variants do not recognize certain N-termini and thus, no N-terminal protection is required. 5. Omniligase-1 has a broad substrate scope and enables the efficient ligation of different peptide fragments. However, some substrates contain suboptimal sequences, which can hamper successful ligation. In order to choose an optimal ligation strategy the user should choose the coupling position mainly based on the following guidance (see Figs. 6 and 7). (a) Try to ensure that the amino acid in P4 position is hydrophobic (preferred) or is one of the following (suboptimal) amino acids: Ser, Thr, or Cys. (b) Try to choose a coupling position in such a way that the amino acid in P20 position has a large hydrophobic side chain. (c) Check if the amino acids in all remaining positions are tolerated. A few suboptimal amino acids can be tolerated and compensated by the addition of more enzyme or longer reaction times. However, the combination of several suboptimal amino acids prevents successful ligation. 6. The presence of several suboptimal amino acids in the recognition sequence might lead to a drastically decreased ligation efficiency. The amino acids in positions P1 to P4 mainly determine the reaction rate and suboptimal amino acids in these positions can often be compensated with the addition of (10or 100-fold) more enzyme. In contrast, with suboptimal amino acids in position P10 and/or P20 no product formation will be observed (only hydrolysis). In most cases, the acyl donor sequence mainly determines the reaction rate and the reaction

Fig. 6 Overview map of omniligase-1 acyl acceptor (P4–P1) and acyl donor (P10 , P20 ) substrate preferences. Please note that the colors indicated only represent a rough guideline based on screening model substrates. Individual acceptance of amino acids in each pocket can vary from peptide to peptide

Omniligase-1-Mediated Peptide Ligation and Cyclization

57

Fig. 7 The acyl acceptor (P10 , P20 ) substrate profile of omniligase-1. For the acyl acceptor site, the S10 and S20 pockets were mapped. All 400 possible combinations of the first two amino acids (proteinogenic) in the substrate positions P10 and P20 (H-Xxx-Yyy-Peptide; 20 mM) were tested together with Ac-DFSKL-OCamL-OH (10 mM in 1 M potassium phosphate buffer, 70 nM omniligase-1). Reaction outcomes (HPLC-% product formation) after 30 min are given, with green being optimal and red impossible. In order to highlight differences between the substrates suboptimal reaction conditions were used for evaluation

yield is depending on the P10 and P20 amino acids of the acyl acceptor fragment. 7. Difficulties with some suboptimal amino acids in certain positions (e.g., Asp in P1 position) can be overcome by using other, substrate-tailored peptiligase variants (e.g., thymoligase) [15]. 8. If peptides differ from given sequences, or fragments are present in different salt form, please ensure to use the molecular weight of your peptide as the corresponding salt form. 9. The peptide concentration is critical for the ligation yield and efficiency. Although ligations work from substrate concentrations in the μM range up to mM concentrations, both intermolecular peptide–peptide as well as peptide–protein ligations should be performed with as concentrated substrates as possible to ensure optimal ligation yields. On the contrary, for

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cyclization reactions, the concentration should remain