Copper-Free Click Chemistry Antibody-DNA Conjugation

Typically, there are three biological functional groups on the peptide for the further conjugation: amino ( –NH2 ), carboxyl ( –COOH), and thiol ( –SH). The most effective way is to utilize the free thiol groups from cysteine. The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules.

The click chemistry is another efficient method to conjugate the peptide with other biomolecules. The peptide can be modified with azide groups (–N3). The novel Copper-free Click Chemistry is based on the reaction of a diarylcyclooctyne moiety (DBCO) with an azide-peptide reaction partner. This click reaction is very fast at room temperature and does not require a cytotoxic Cu(I) catalyst, resulting in almost quantitative yields of stable triazoles. The DBCO allows Copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. The reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (–SH, thiol).

One example of the peptide drug conjugations is the antibody-biomolecule conjugate.

click chemistry: DBCO-azide

click chemistry: DBCO-azide

A simple protocol: Click chemistry of antibody-DNA conjugation

Pre-conjugation considerations

  • Remove all additives from antibody solutions using dialysis or desalting.
  • Remove BSA and gelatin from antibody solutions.
  • Concentrate the antibody after dialysis or purification.

Activation of antibodies with DBCO-NHS ester

  • Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
  • Incubates at room temperature for 30 min or 2 hours on ice.

Quenching activation reaction

  • Add Tis-Hcl (50-100mM, pH 8) to the reaction.
  • Incubate at RT for 5 min or 15 minutes on ice.

Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction)

Copper-Free click reaction

  • Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
  • Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.

Validation of conjugation and purification by HPLC

Selected References:

  1. Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
  2. Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst-free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
  3. Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.

A Simple Protocol to Refold Peptides or Small Proteins

Understanding the process of peptide folding is a critical first step toward understanding protein folding. Depending on the temperature and solvent conditions, peptides are highly flexible and can adopt a variety of conformations in solution. Many unfolded peptides could spontaneously refold in vitro to form a native protein with full biological activity in the absence of other factors.

The primary sequence contains all the information to define the three dimensional structure of a protein and its biological functions. The mutation or deletion of any amino acid may have a big impact on folding and stability. It takes nanoseconds (ns) for the peptide to form an intermolecular contact. The timescales of loop closing is 10 nanosecond (ns). The formation of alpha-helical peptides is 200 ns,  beta hairpins and mini-proteins in 1–10 ms timescale. Many studies had a very good agreement between measured and calculated folding rates. Many factors such as temperature, pH,  molecular chaperones, salts, and denaturant may affect a peptide in reaching its native state.

So it is critical to minimize factors that affect protein refolding. A successful folding should have inadequate denaturant concentrations to destabilize the native state of a peptide or protein. GuHCL can be used in order to disrupt the hydrophobic interactions within the tertiary structure.

  1. The peptide was solubilized in resuspension buffer (50 mM Tris, pH 8, 6 M GuHCl (Sigma, G4505), 10 mM DTT, 2mM EDTA) by vortexing.
  2. Use enough resuspension buffer such that the final peptide concentration is 0.2mg/ml.
  3. The resuspended peptide was then diluted 50% in dialysis buffer #1 (50 mM Tris, pH 8, 2 M GuHCl, 2mM EDTA) resulting in a 4 M GuHCl containing solution.
  4. The peptide solution was then dialyzed overnight at 4°C in snakeskin dialysis tubing (Pierce) against 2 L of buffer #1.
  5. The following day the dialysis buffer was changed to 2 L of dialysis buffer #2 (50 mM Tris, pH 8, 1 M GuHCl, 0.4 M Arginine (Sigma, A5006), 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione, 2mM EDTA) for overnight dialysis at 4°C.
  6. The following day the dialysis buffer was diluted 50% with water and dialysis continued overnight.
  7. Any insoluble material was centrifuged (18000×g at 2–8°C for 20 minutes) and the remaining peptide solution dialyzed overnight at 4°C against 1 L of dialysis buffer #3 (50 mM Tris, pH 8, 250 mM NaCl, 0.1 M Arginine, 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione, 2mM EDTA) to remove the remaining GuHCl.
  8. The final dialyzed protein solution was clarified by centrifugation (18000×g at 2–8°C for 20 minutes) and the supernatant separated by RP-HPLC.

References:

Synthetic Peptides Used for indirect ELISA

ELISA

Peptides can be used for ELISA assay. A peptide-based indirect ELISA was used to screen a population of 40 Multiple sclerosis patients and 39 healthy controls. The encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35–55 synthetic peptides were synthesized by LifeTein.

All MOG peptides or Mycobacterium avium subspecies paratuberculosis (MAP) peptides were synthesized at >90% purity by LifeTein to make sure the ELISA results are clean and consistent. The plates were coated with peptides. After overnight incubation, the plate was blocked, rinsed, and late react with antibodies according to the protocol. In silico analysis identified two peptides belonging to MAP and BCG, which share sequence homology with MOG(35–55). The peptide-based indirect ELISA data showed that sharing of highly conserved linear amino acidic sequences is necessary to elicit antibody-mediated cross-reactivity.

These findings concluded that the presence of MOG (35–55)-specific antibodies in multiple sclerosis pathogenesis. This can be used as a diagnostic biomarker in multiple sclerosis.

 [PDF] Evaluation of the humoral response against mycobacterial peptides, homologous to MOG35–55, in multiple sclerosis patients

MG Marrosu, LA Sechi – 2014

 All peptides were synthesized at N90% purity commercially (LifeTein, South
Plainfield, NJ 07080 US). Purified peptides were prepared as [10 mM] stock solutions,
and were stored in single- use aliquots at −80 °C. 2.3. ELISA