Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

• Publication year

  2018

• Venue

  Journal of the American Chemical Society

• Publication date

  2018-06-21

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1021/jacs.8b05200PMID 29927249PMCID PMC6230430
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

  2018cited by this paper
• The "Lid" in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site.

  2016cited by this paper
• Site-selective protein-modification chemistry for basic biology and drug development.

  2016cited by this paper
• Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent

  2016cited by this paper
• CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

  2016cited by this paper
• Recent advances in sortase-catalyzed ligation methodology.

  2016cited by this paper
• Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher.

  2015cited by this paper
• Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency

  2015cited by this paper
• Enzymatic bioconjugation of nanoparticles: developing specificity and control.

  2015cited by this paper
• A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

  2014cited by this paper
• Reprogramming the specificity of sortase enzymes

  2014cited by this paper
• Selective chemical protein modification

  2014cited by this paper
• Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

  2014cited by this paper
• Enzymatic labeling of proteins: techniques and approaches.

  2013cited by this paper
• Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro

  2013cited by this paper
• Pili of gram-positive bacteria: roles in host colonization.

  2012cited by this paper
• Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

  2012cited by this paper
• Efficient N-terminal labeling of proteins by use of sortase.

  2012cited by this paper
• Isopeptide Ligation Catalyzed by Quintessential Sortase A

  2011cited by this paper
• A general strategy for the evolution of bond-forming enzymes using yeast display

  2011cited by this paper
• Protein-Protein Fusion Catalyzed by Sortase A

  2011cited by this paper
• Sortase enzymes in Gram‐positive bacteria

  2011cited by this paper
• Structure of the sortase AcSrtC-1 from Actinomyces oris.

  2011cited by this paper
• In vitro Sortagging of an Antibody Fab Fragment: Overcoming Unproductive Reactions of Sortase with Water and Lysine Side Chains

  2011cited by this paper
• Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

  2009cited by this paper
• Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

  2009cited by this paper
• Sortase activity is controlled by a flexible lid in the pilus biogenesis mechanism of gram-positive pathogens.

  2009cited by this paper
• Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation

  2009cited by this paper
• Sortase-mediated pilus fiber biogenesis in Streptococcus pneumoniae.

  2008cited by this paper
• Lipid modification of proteins through sortase-catalyzed transpeptidation.

  2008cited by this paper
• Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase.

  2008cited by this paper
• Peptide-sugar ligation catalyzed by transpeptidase sortase: a facile approach to neoglycoconjugate synthesis.

  2008cited by this paper
• How Some Pili Pull

  2007cited by this paper
• Biochemistry. How some pili pull.

  2007cited by this paper
• Sortagging: a versatile method for protein labeling.

  2007cited by this paper
• Assembly of pili in Gram-positive bacteria.

  2004cited by this paper
• Properties and applications of microbial transglutaminase

  2004cited by this paper
• Sortase-mediated protein ligation: a new method for protein engineering.

  2004cited by this paper
• Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall.

  1999cited by this paper

• Stepwise dual N-terminal modification of proteins via nickel(II)-mediated aldol and Pictet–Spengler reactions

  2026cites this paper
• OaAEP1-dependent enzymatic protein ligation and immobilization for single-molecule force spectroscopy.

  2025cites this paper
• Semi-synthesis of functionalized cell-permeable ubiquitin probes via SrtA-mediated ligation.

  2025cites this paper
• Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.

  2024cites this paper
• Site-specific protein labeling: Recent progress

  2024cites this paper
• Repurposing Amide Bond-Forming Enzymes for Non-native Protein Modification

  2024cites this paper
• Sequence‐Dependent Acylation of Peptide Lysine Residues by DNAzymes

  2024cites this paper
• Sortases: structure, mechanism, and implications for protein engineering.

  2024cites this paper
• Recent Toolboxes for Chemoselective Dual Modifications of Proteins.

  2024cites this paper
• Biogenesis and Functionality of Sortase-Assembled Pili in Gram-Positive Bacteria.

  2024cites this paper
• The Basal and Major Pilins in the Corynebacterium diphtheriae SpaA Pilus Adopt Similar Structures that Competitively React with the Pilin Polymerase

  2023cites this paper
• Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation.

  2023cites this paper
• Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

  2023cites this paper
• Accelerating the design of pili-enabled living materials using an integrative technological workflow

  2023cites this paper
• Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43

  2022cites this paper
• Challenges in the use of sortase and other peptide ligases for site-specific protein modification

  2022cites this paper
• Thioester-Assisted Sortase-A - Mediated Ligation.

  2022cites this paper
• Amyloid fibrils in frontotemporal lobar degeneration with TDP-43 inclusions are composed of TMEM106B, rather than TDP-43

  2022cites this paper
• A review on pilus assembly mechanisms in Gram-positive and Gram-negative bacteria

  2022cites this paper
• Site-specific internal protein labeling through trans-splicing.

  2021cites this paper
• Anchoring surface proteins to the bacterial cell wall by sortase enzymes: how it started and what we know now.

  2021cites this paper
• Enzymatic Protein-Protein Conjugation through Internal Site Verified at the Single-Molecule Level.

  2021cites this paper
• Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits

  2021cites this paper
• Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism

  2021cites this paper
• A Fast, Efficient and Easy to Implement Method to Purify Bacterial Pili From Lacticaseibacillus rhamnosus GG Based on Multimodal Chromatography

  2020cites this paper
• Syntheses of Bromo-N-Heterocycles through DBH-Promoted Tandem C-H Amination/Bromination.

  2020cites this paper
• Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials

  2020cites this paper
• Column-free purification and coating of SpyCatcher protein on ELISA wells generates universal solid support for capturing of SpyTag-fusion protein from the non-purified condition

  2020cites this paper
• Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

  2020cites this paper
• New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria.

  2020cites this paper
• The proteasome-related Connectase is a fast and specific protein ligase

  2020cites this paper
• Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins

  2020cites this paper
• DNAzymes for amine and peptide lysine acylation.

  2020cites this paper
• A novel full symmetric diarylethene-based ratiometric fluorescent sensor for lysine and the application for logic circuit.

  2020cites this paper
• Engineered Sortases in Peptide and Protein Chemistry

  2020cites this paper
• Lysine Acylation Using Conjugating Enzymes (LACE) for Site-Specific Modification and Ubiquitination of Native Proteins

  2019cites this paper
• Dynamic control of hydrogel crosslinking via sortase-mediated reversible transpeptidation.

  2019cites this paper
• Isopeptide Ligations Catalyzed by Streptococcus Suis Sortase A

  2019cites this paper
• Approaching infinite affinity through engineering of peptide–protein interaction

  2019cites this paper
• Amide-Forming Ligation Reactions

  2019cites this paper
• Broadening the scope of sortagging

  2019cites this paper