Figure 2Īnalysis of Table 2 shows that the biggest shift in the value of the calculated pI (decrease by 0.6) is observed for profilins, which is expected as these proteins are the smallest among those studied here, and the His-tag corresponds to a significant fraction of the polypeptide chain ( Table 1). Bet v 2 was most stable in the pH range 8.5–9.5, and the His-tag increased its stability by ∼4 ☌. Art v 4 was most stable in the pH range 8.0–9.5, and the His-tag did not significantly impact stability. Amb a 8 was most stable in the pH range 8.5–9.5, and the His-tag slightly increased its stability. NgDapB was the most stable in the pH range 8.0–9.0, and the His-tag did not impact stability except at lower pH values (5.0–6.0), where His-NgDapB was less stable. Furthermore, His-MtDapB was very unstable in the pH range 5.0–6.0, whereas MtDapB showed much higher stability (∼20 ☌). MtDapB was most stable in the pH range 8.5–9.5, and His-MtDapB was less stable in this same range by ∼4 ☌. McsA was most stable in the pH range 6.0–7.0 and the His-tag did not significantly impact stability. VvDxr was most stable in the pH range 5.5–6.5 with His-VvDxr being slightly more stable under these conditions than VvDxr. SpNadD was most stable in the pH range 8.5–9.5, and the His-tag did not affect stability based on pH. β-Lactamase was most stable in the pH range 6.0–7.0 and without the His-tag. (26-28)įigure 2 shows DSF results for all proteins in the presence of sodium chloride along with varied pH, whereas Table 2 summarizes the highest melting temperatures and best buffering conditions for proteins studied. (19-25) Additionally, DSF can also be used as a rapid screening method for finding protein ligands and/or compounds that stabilize proteins. (18) Such an approach is a low-cost method to obtain high-throughput thermal scanning results and has been proven to be very effective in the optimization of the thermal stability of proteins. These experiments are performed using a real-time polymerase chain reaction (RT-PCR) instrument that is compatible with small volumes and capable of reaching a broad temperature range. T m is the inflection point of the resulting fluorescence curve and serves as a proxy for the stability of a protein sample. As the mixture is gradually heated over time, the protein starts to denature and inner hydrophobic residues are exposed the fluorescent dye binds to the exposed hydrophobic fragments, and the melting temperature ( T m) of the protein can be determined by monitoring fluorescence over time. (17) In this method, samples of protein are mixed with different solutions (containing, for example, ligands, buffer solutions, and salt conditions) and a fluorescent hydrophobic dye. DSF, also known as thermal-shift fluorescence-based screening or Thermofluor, is a cost-effective and rapid screening method that may be used to study protein stability or ligand-binding properties. In this study, differential scanning fluorimetry (DSF) was used to determine the effect of the N-terminal His-tag on protein stability. (13) Moreover, His-tags may also have an impact on structural studies and thus the ability to crystallize proteins for X-ray diffraction studies. (8, 9, 12) In the case of sperm whale myoglobin, the addition of an N-terminal His-tag affected its conformational movements, which indicates that the His-tag should be taken into account during the investigation of protein structural dynamics. (8, 9, 12-15) Significant reductions in the enzymatic activity of several different enzymes were observed upon the incorporation of a His-tag. (11) His-tags may affect the oligomeric states of proteins as well as their function. demonstrated the effects of varying the length and position of His-tags on the expression and production of a membrane protein.
(5, 6) However, there is an increase in the number of reports showing that this tacit assumption may be false.
His-tags, due to their relatively small size (∼2.5 kDa), are not believed to significantly interfere with the function and structure of a majority of proteins.