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.

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