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

Booth, William T.; Schlachter, Caleb R.; Pote, Swanandi; Ussin, Nikita K.; Mank, Nicholas; Klapper, V.; Offermann, L.R.; Tang, Chuanbing; Hurlburt, Barry K.; Chruszcz, M.

doi:10.1021/acsomega.7b01598

Cited by 140 publications

(75 citation statements)

References 45 publications

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“…Although our collection facilitates tagging of SARS-CoV-2 proteins for various functional studies, certain applications require removal of tags at some stage, for example, after protein purification. Fusion proteins can potentially interfere with the yield, structure, and function of purified proteins, such as during large scale production and crystallography studies (Booth et al 2018). To address this we expanded our collection to include clones containing an N-terminal recognition sequence for the nuclear inclusion protease from tobacco etch virus (TEV; Carrington and Dougherty 1987;Carrington and Dougherty 1988).…”

Section: Resultsmentioning

confidence: 99%

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions

Kim

Knapp

Kuang

et al. 2020

G3 Genes|Genomes|Genetics

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The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.

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Section: Resultsmentioning

confidence: 99%

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions

Kim

Knapp

Kuang

et al. 2020

G3 Genes|Genomes|Genetics

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“…The observed 2.5‐fold reduction in the catalytic activity of the AtcTPI‐Cys218Ser mutant contrasts with the 142‐fold reduction previously reported using an AtcTPI with a large non‐related amino acid sequence in its N‐terminal (López‐Castillo et al ., ). This differential in activity correlates with the presence a long sequence tag in the previous constructs that may hamper their activity (Panek et al ., ; Sabaty et al ., ; Booth et al ., ).…”

Section: Resultsmentioning

confidence: 97%

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

et al. 2019

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Summary Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

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“…In most cases, the incorporation of His-tag has resulted in great increases in the efficiency of the protein purification process however, sometimes, it hampers the protein functionality. Then, several experiments were conducted to remove the additional histidine residues but drawbacks remain if nonwild type amino acids remain in the protein 18 . In our experiments we evaluate the enzymatic activity of the wild type β-GalWT, the commercial enzyme from Sigma, and a recombinant β-GalHis that we produced and purified by IMAC chromatography.…”

Section: β-Gals Functionalitymentioning

confidence: 99%

“…It is usually accepted that due to their small size, His-tags would not interfere with the function and structure of the majority of proteins 10,15 . However, there is an increase number of reports showing that this conception may be not always true [16][17][18] . Moreover, there are evidences that His-tags may affect the oligomeric states of proteins as well as their function [19][20][21] In the present study we compared a β-Gal containing a hexahistidine peptide added in the Cterminal, β-GalHis, with its wild type commercial counterpart, β-GalWT.…”

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confidence: 99%

Advantages and Disadvantages of His-Tagged Beta-Galactosidase

Flores

Clop

Barra

et al. 2020

Preprint

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β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific.

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

Cited by 140 publications

References 45 publications

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Advantages and Disadvantages of His-Tagged Beta-Galactosidase

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