Tagging/bioconjugation techniques

• An isopeptide bond is an amide bond formed between a carboxyl/amide group of one amino acid and the amino group of another.
• Isopeptide bonds form to stabilise proteins in gram-positive bacteria because they lack the periplasmic space, which provides an oxidising environment for the formation of disulfide bonds.
• Some examples of proteins that contain isopeptide bonds:

  • Proteins found in gram-positive bacteria
  • Ubiquitin/ubiquitin ligase
  • Transglutaminase
  • Complement proteins
  • Alpha2-macroglobulins
• The three residues involved in isopeptide bond formation in the protein core:

  • Lysine (low pKa so good nucleophile)
  • Glutamic acid (polarises Asn/Asp side chain)
  • Asparagine/aspartic acid (undergoes the attack)
• SpyTag/SpyCatcher system


  Exploits isopeptide bond formation to identify protein-ligand interactions
• SpyCatcher


  • 15 kDa protein engineered from CnaB2 domain of FbaB protein in Streptococcus pyogenes
  • Contains Lys31 residue - forms irreversible isopeptide bond with SpyTag (13 amino acid peptide domain AHIVMVDAYKPTK)
• Using the SpyTag/SpyCatcher system

  1. Fuse protein to SpyCatcher

  2. Fuse ligand to SpyTag (gene constructs, express)

  3. Binding of protein and ligand forces SpyCatcher and SpyTag into binding proximity

  4. Irreversible isopeptide bond formation
• Detecting interaction (i.e. complex formation)

  Using gel electrophoresis, Western blot etc
• Thioester intermediate forms during peptide/isopeptide bond formation. Thioester reacts with primary amine to form amide (peptide) bond and thiol
• Isopeptide bonds in gram-positive bacteria are exploited to link between the bacteria surface and host cells.

  E.g. between Q261 in thioester Q/C form in Sfbl protein in S. pyogens and K100 in fibrinogen A
• Sortase is an enzyme expressed in gram-positive bacteria that catalyses the formation of a peptide bond between secreted proteins with the LPXTG motif and peptidoglycans, to anchor the secreted protein in the peptidoglycan so its domains can be exploited to better anchor and attack the host cell.
• Sortase


  Enzyme engineered to remove the domain that anchors it to the surface of gram-positive bacteria, making it soluble
• Exploiting sortase for protein engineering

  1. Engineer protein of interest to contain LPXTG motif (acyl donor)

  2. Recombinantly express the construct

  3. Modify target protein to contain sortase recognition motif (acyl acceptor)

  4. Incubate engineered sortase and acyl donor construct (sortase cleaves LPXTG, leaving thioester intermediate)

  5. Add acyl acceptor construct (acyl acceptor performs nucleophilic attack on acyl donor, forming peptide bond, sortase released)
• Sortase tagging can be used to tag red blood cells with additional protein domains - e.g. a drug for targeted drug delivery or immunotherapy
• Bioorthogonal chemistry works selectively in biological environments without side reactions
• Proteins are difficult to modify as typically involves extremes of pH and temperature, but at these extremes, proteins denature and aggregate
• NHS esters are used to tag proteins.

  NHS ester + primary amine (protein) -> stable conjugate (protein and tag joined by amide bond) + NHS
• To successfully tag proteins via NHS esters:

  • Carry out reactions at pH 7.2 - 8.5 at room temperature
  • Exclude buffers containing primary amines e.g. Tris
  • Use Tris or glycine buffer to stop the reaction
• Using NHS esters to tag proteins is advantageous as the hydrophobic cross-linker means the tag can cross membranes
• An example of using NHS esters to tag a protein: biotinylation of a protein to permit protein-ligand interaction to be identified via the detection of the biotin-avidin complex
• Maleimides are used to modify cysteine residues
• Maleimides modify cysteines by:

  • Lone pair on thiol on cysteine performs nucleophilic attack on beta carbon of maleimide
  • Imide nitrogen acts as a leaving group, resulting in thioether bond formation
• Beta-mercaptoethanol and TCEP should be excluded from assays with stable thioester conjugates (i.e. those formed from maleimides) as they compete for coupling sites
• Photo-reactive reagents are chemically inert - reactive when exposed to UV or visible light; proteins modified with photoaffinity labeling (PAL) reagents can covalently bind their targets after activation by light
• Photoaffinity labelling can label low abundance and low affinity proteins
• Ideal properties of PAL reagents:

  • Stable in the dark in a range of pHs
  • Activation at wavelengths that do minimal damage to biomolecules
  • Still capable to generate intermediates that react and form stable adducts
  • Minimal side reactions
• There are three important functionalities of PAL reagents:

  • Moiety is photoreactive
  • Specificity unit can bind to target protein and is cleavable
  • Reporter tag e.g. fluorescent dye, radioisotope, to identify binding event