Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity.

ABSTRACT: The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.

SUBMITTER: Turkewitz DR

PROVIDER: S-EPMC8138732 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Rapid labeling of intracellular His-tagged proteins in living cells.

Project description:Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

| S-EPMC4364221 | biostudies-literature

Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins.

Project description:To date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging.Four different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, ?-CD33 scFv, ?-VCAM-1 scFv and ?-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins.We used 260 and 350 ?L [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+].We have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.

| S-EPMC4015829 | biostudies-other

Bound-state residual dipolar couplings for rapidly exchanging ligands of His-tagged proteins.

Project description:The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound-state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.

| S-EPMC2542485 | biostudies-literature

Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess.

Project description:With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O2, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the Brevibacillus culture media.

| S-EPMC8347774 | biostudies-literature

Intracellular Delivery of His-Tagged Genome-Editing Proteins Enabled by Nitrilotriacetic Acid-Containing Lipidoid Nanoparticles.

Project description:Protein- and peptide-based therapeutics with high tolerance and specificity along with low off-target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His-tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His-tagged proteins including green fluorescent protein (GFP), (-30)GFP-Cre recombinase, and CRISPR/Cas9 ribonucleoprotein into mammalian cells.

| S-EPMC6474682 | biostudies-literature

A Simple and Versatile Strategy for Oriented Immobilization of His-Tagged Proteins on Magnetic Nanoparticles.

Project description:Oriented and covalent immobilization of proteins on magnetic nanoparticles (MNPs) is particularly challenging as it requires both the functionality of the protein and the colloidal stability of the MNPs to be preserved. Here, we describe a simple, straightforward, and efficient strategy for MNP functionalization with proteins using metal affinity binding. Our method involves a single-step process where MNPs are functionalized using a preformed, ready-to-use nitrilotriacetic acid-divalent metal cation (NTA-M2+) complex and polyethylene glycol (PEG) molecules. As a proof-of-concept, we demonstrate the oriented immobilization of a recombinant cadherin fragment engineered with a hexahistidine tag (6His-tag) onto the MNPs. Our developed methodology is simple and direct, enabling the oriented bioconjugation of His-tagged cadherins to MNPs while preserving protein functionality and the colloidal stability of the MNPs, and could be extended to other proteins expressing a polyhistidine tag. When compared to the traditional method where NTA is first conjugated to the MNPs and afterward free metal ions are added to form the complex, this novel strategy results in a higher functionalization efficiency while avoiding MNP aggregation. Additionally, our method allows for covalent bonding of the cadherin fragments to the MNP surface while preserving functionality, making it highly versatile. Finally, our strategy not only ensures the correct orientation of the protein fragments on the MNPs but also allows for the precise control of their density. This feature enables the selective targeting of E-cadherin-expressing cells only when MNPs are decorated with a high density of cadherin fragments.

| S-EPMC10739578 | biostudies-literature

Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii.

Project description:Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6xHis tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.

| S-EPMC2838008 | biostudies-literature

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli.

Project description:Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure.

| S-EPMC3660886 | biostudies-literature

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus.

Project description:Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

| S-EPMC7126649 | biostudies-literature

The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'.

Project description:The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag-bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one-pot purifications and in cases where tag removal is difficult.

| S-EPMC9716568 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data