Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

John M. Antos1, Jessica Ingram2, Tao Fang3, Novalia Pishesha4, Matthias C. Truttmann3, Hidde L. Ploegh3

1 Department of Chemistry, Western Washington University, Bellingham, Washington, 2 Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, 3 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 15.3
DOI:  10.1002/cpps.38
Online Posting Date:  August, 2017
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Abstract

Strategies for site‐specific protein modification are highly desirable for the construction of conjugates containing non‐genetically‐encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non‐natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five‐amino‐acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N‐ or C‐terminus in site‐specific fashion. As described in this unit, the successful implementation of sortase‐mediated labeling involves straightforward solid‐phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.

Keywords: sortase; transpeptidation; site‐specific labeling; chemoenzymatic labeling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Site‐Specific Labeling of Purified Proteins via Sortase‐Mediated Transpeptidation (N‐ or C‐Terminal Labeling)
  • Alternate Protocol 1: Labeling Cell‐Surface Proteins in Living Cells via Sortase‐Mediated Transpeptidation
  • Support Protocol 1: Expression and Purification of SrtAstaph Pentamutant and Heptamutant
  • Support Protocol 2: Synthesis of Oligoglycine Probes for C‐Terminal Labeling and LPXTG Probes for N‐Terminal Labeling
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Site‐Specific Labeling of Purified Proteins via Sortase‐Mediated Transpeptidation (N‐ or C‐Terminal Labeling)

  Materials
  • Purified LPXTG‐containing target protein (see Strategic Planning; cannot be in phosphate‐containing buffer when using pentamutant variant of sortase)
  • Purified sortase A pentamutant or heptamutant stock solution (see Strategic Planning and protocol 3)
  • Oligoglycine peptide probe stock solution (5 to 10 mM in DMSO or H 2O; protocol 4)
  • 10× sortase reaction buffer:
    • for pentamutant: 500 mM Tris·Cl, pH 7.5 ( appendix 2E), 1.5 M NaCl, 100 mM CaCl 2)
    • for heptamutant: 500 mM Tris·Cl, pH 7.5 ( appendix 2E) supplemented with 1.5 M NaCl, or 10× phosphate buffered saline (PBS; appendix 2E)
    • Purified target protein containing 1 to 5 N‐terminal glycine residues (see Strategic Planning; cannot be in phosphate‐containing buffer when using pentamutant variant of sortase)
    • LPXTGG peptide probe stock solution (5 to 10 mM in DMSO or H 2O; protocol 4)
    • Ni‐NTA column (optional; see unit 9.4; Petty, )
    • Imidazole (optional)
  • 1.5‐ml microcentrifuge tubes
  • 4°C or 25°C incubator
  • Zeba desalting column (ThermoFisher) or centrifugal concentrator with MWCO below the molecular weight of the target protein (Millipore)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, ), Coomassie staining (unit 10.5; Echan & Speicher, ), immunoblotting (unit 10.1; Ni, Xu, & Gallagher, ), mass spectroscopy (see Chapter 16 in this manual), and Ni‐NTA chromatography (unit 9.4; Petty, )

Alternate Protocol 1: Labeling Cell‐Surface Proteins in Living Cells via Sortase‐Mediated Transpeptidation

  Materials
  • Target cells
  • Plasmid encoding target protein (type II membrane protein with C‐terminal LPXTG motif or type I membrane protein with 1 to 5 N‐terminal glycines)
  • Transfection reagent (Lipofectamine, Invitrogen; Trans IT, Mirus; FuGENE 6, Promega)
  • Culture medium such as DMEM (phenol red–free; presence of 10% serum does not inhibit the sortase reaction)
  • 1 mM purified sortase A heptamutant stock solution ( protocol 3)
  • 10 mM peptide probe stock in DMSO or H 2O (oligoglycine probe for labeling type II membrane proteins, LPXTGG probe for labeling type I membrane proteins; protocol 4)
  • 10× sortase reaction buffer: 500 mM Tris·Cl, pH 7.5 ( appendix 2E) containing 1.5 M NaCl
  • Phosphate‐buffered saline (PBS) (without Ca2+ or Mg2+; appendix 2E)
  • 1.5‐ml microcentrifuge tubes
  • Plastic tissue culture dishes
  • Microscope

Support Protocol 1: Expression and Purification of SrtAstaph Pentamutant and Heptamutant

  Materials
  • Sortase pentamutant or heptamutants expression plasmid: pET30b (available from Addgene or via request from the authors)
  • E. coli BL‐21(DE3) competent cells (New England Biolabs)
  • Terrific broth (TB) medium with and without appropriate antibiotic: kanamycin (1000× stock = 30 mg/ml in H 2O)
  • LB agar plates containing 30 μg/ml kanamycin
  • 1 M isopropyl β‐D‐thiogalactopyranoside (IPTG) in H 2O
  • Lysis buffer: 50 mM Tris·Cl, pH 8 ( appendix 2E), 150 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol
  • 10 mg/ml DNAse I stock solution in H 2O
  • Ni‐NTA agarose slurry (Qiagen)
  • Elution buffer: 50 mM Tris·Cl, pH 8 ( appendix 2E), 150 mM NaCl, 10% (v/v) glycerol (v/v), with and without 350 mM imidazole
  • Culture plates and tubes
  • 4‐liter culture flask
  • 37° and 30°C incubators with shaking
  • Spectrophotometer and appropriate cuvettes
  • Refrigerated centrifuge
  • French press (pre‐chilled; see unit 6.2; Wingfield, ) or probe sonicator (Branson Sonifier 450)
  • 1.5 × 12–cm disposable polypropylene column (BioRad)
  • Centrifugal concentrator with low <10 kD MWCO (optional, Millipore)
  • Additional reagents and equipment for Ni‐NTA chromatography (unit 9.4; Petty, )

Support Protocol 2: Synthesis of Oligoglycine Probes for C‐Terminal Labeling and LPXTG Probes for N‐Terminal Labeling

  Materials
  • Rink amide resin, 100 to 200 mesh, cross‐linked with 1% (w/w) divinylbenzene (Advanced ChemTech, cat. no. SA5030)
  • Dichloromethane (DCM)
  • Piperidine
  • N,N‐dimethylformamide (DMF)
  • Fmoc protected amino acids:
    • Fmoc‐Gly 3‐OH (ChemImpex, cat. no. 08072)
    • Fmoc‐Cys(Trt)‐OH (Novabiochem, cat. no. 852008)
    • Fmoc‐Lys(Mtt)‐OH (Novabiochem, cat. no. 852065)
    • Fmoc‐Leu‐OH (Novabiochem, cat. no. 852011)
    • Fmoc‐Pro‐OH (Novabiochem, cat. no. 852017)
    • Fmoc‐Glu(tBu)‐OH (Novabiochem, cat. no. 852009)
    • Fmoc‐Thr(tBu)‐OH (Novabiochem, cat. no. 852000)
    • Fmoc‐Gly‐OH (Novabiochem, cat. no. 852001)
  • HBTU (2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexauorophosphate; ChemPep, cat. no. 120802)
  • Diisopropylethylamine (DIPEA)
  • Trifluoroacetic acid (TFA)
  • Triisopropylsilane (TIPS)
  • 5(6)‐carboxytetramethylrhodamine (TAMRA)
  • Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)
  • Diethyl ether, ice cold
  • Phosphate buffered saline (without Ca2+ or Mg2+; appendix 2E)
  • Maleimide‐ or NHS‐derived modification (Conju‐probe, ThermoFisher, or Lumiprobe)
  • Dimethylsulfoxide (DMSO)
  • EDT (1,2‐ethanedithiol)
  • Solid‐phase peptide synthesis vessel with associated caps and filter frits
  • Wrist‐action shaker or similar equipment for mixing solid‐phase reaction vessels
  • Vacuum source
  • HPLC system with C18 reversed‐phase column (unit 8.7; Josic & Kovac, )
  • Lyophilizer
  • Additional reagents and equipment for reversed‐phase HPLC (unit 8.7; Josic & Kovac, ) and mass spectroscopy (Chapter 16)
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Figures

Videos

Literature Cited

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