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. 2018 Jan 31;3(1):760-768.
doi: 10.1021/acsomega.7b01598. Epub 2018 Jan 22.

Impact of an N-terminal Polyhistidine Tag on Protein Thermal Stability

William T Booth  1 , Caleb R Schlachter  1 , Swanandi Pote  1 , Nikita Ussin  1 , Nicholas J Mank  1 , Vincent Klapper  1 , Lesa R Offermann  1   2 , Chuanbing Tang  1 , Barry K Hurlburt  3 , Maksymilian Chruszcz  1
Affiliations

Impact of an N-terminal Polyhistidine Tag on Protein Thermal Stability

William T Booth et al. ACS Omega. .

Abstract

For years, the use of polyhistidine tags (His-tags) has been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a consequence of His-tag addition is that it can affect protein function and stability. Functional proteins are essential in the elucidation of their biological, kinetic, structural, and thermodynamic properties. In this study, we determine the effect of N-terminal His-tags on the thermal stability of select proteins using differential scanning fluorimetry and identify that the removal of the His-tag can have both beneficial and deleterious effects on their stability.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Examples of screenings from DSF. A gradient of pH (4.0–9.5) and salt (0.0–1.0 M NaCl) buffers were used. Red, white, and green represent low, average, and high melting temperatures (°C), respectively. The standard deviation was typically less than 1 °C for all experiments. (A) Data for His-β-lactamase and β-lactamase. (B) Data for His-Bet v 2 and Bet v 2. (C) Data for His-MtDapB and MtDapB.
Figure 2
Figure 2
Effect of pH and His-tag on protein stability. Only melting temperatures observed from solutions with no salt are shown. Red, white, and green represent low, average, and high melting temperatures, respectively. All temperatures are in degree Celsius.
Figure 3
Figure 3
Effect of salt concentration and His-tag on protein stability. Melting temperatures are shown for solutions containing no salt and 1.0 M salt. The pH of each solution was the pH that displayed the best thermal stability for each respective protein.
Figure 4
Figure 4
Cartoon representations of proteins analyzed in this study with marked positions of their N-termini. (A) Amb a 8 (PDB code: 5EV0) in complex with polyproline (blue). Both Arv 4 and Bet v 2 have the same overall fold as Amb a 8. (B) mcsA (PDB code: 5UQO). (C) Homolog of SpNadD (NadD from Escherichia coli (E. coli); PDB code: 1K4M). (D) β-Lactamase from Staphylococcus aureus (PDB code: 3BLM). (E) VvDxr (PDB code: 5KQO). (F) MtDapB (PDB code: 1YL7). Both NgDapB and VvDapB have the same overall fold and quaternary structure as MtDapB.
See this image and copyright information in PMC

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