Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Targeting quantum dots to surface proteins in living cells with biotin ligase.

ABSTRACT: Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

SUBMITTER: Howarth M

PROVIDER: S-EPMC1129026 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification of G-quadruplex-interacting Proteins in Living Cells using an artificial G4-targeting Biotin ligase

Project description:We engineering a G4-targeted biotin ligase probe (G4PID), which consists of three parts, RHAU23 at the C-terminus, miniTurbo at the N-terminus, and a flexible linker in the middle to prevent the disturbance of the two functional moieties. We used eCLIP to verified the G4s affinity of G4PID. We also used eCLIP method to verify the direct G4 interaction of Splicing factor 3B subunit 4 (SF3B4) in cellular transcriptome.

2024-03-07 | GSE223574 | GEO

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase

Project description:G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins.

2024-05-24 | PXD045634 | Pride

Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins.

Project description:Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

| S-EPMC2629800 | biostudies-literature

Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells.

Project description:We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.

| S-EPMC3528850 | biostudies-literature

Identification of G-quadruplex-interacting Proteins in Living Cells using an artificial G4-targeting Biotin ligase

Project description:Identification of G-quadruplex-interacting Proteins in Living Cells using an artificial G4-targeting Biotin ligase

| PRJNA926896 | ENA

HaloTag protein-mediated specific labeling of living cells with quantum dots.

Project description:Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging.

| S-EPMC2553894 | biostudies-literature

Monovalent, reduced-size quantum dots for imaging receptors on living cells.

Project description:We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

| S-EPMC2637151 | biostudies-literature

Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells.

Project description:UnlabelledInternalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1007/s11671-009-9307-9) contains supplementary material, which is available to authorized users.

| S-EPMC2893999 | biostudies-literature

Targeting of embryonic stem cells by peptide-conjugated quantum dots.

Project description:BackgroundTargeting stem cells holds great potential for studying the embryonic stem cell and development of stem cell-based regenerative medicine. Previous studies demonstrated that nanoparticles can serve as a robust platform for gene delivery, non-invasive cell imaging, and manipulation of stem cell differentiation. However specific targeting of embryonic stem cells by peptide-linked nanoparticles has not been reported.Methodology/principal findingsHere, we developed a method for screening peptides that specifically recognize rhesus macaque embryonic stem cells by phage display and used the peptides to facilitate quantum dot targeting of embryonic stem cells. Through a phage display screen, we found phages that displayed an APWHLSSQYSRT peptide showed high affinity and specificity to undifferentiated primate embryonic stem cells in an enzyme-linked immunoabsorbent assay. These results were subsequently confirmed by immunofluorescence microscopy. Additionally, this binding could be completed by the chemically synthesized APWHLSSQYSRT peptide, indicating that the binding capability was specific and conferred by the peptide sequence. Through the ligation of the peptide to CdSe-ZnS core-shell nanocrystals, we were able to, for the first time, target embryonic stem cells through peptide-conjugated quantum dots.Conclusions/significanceThese data demonstrate that our established method of screening for embryonic stem cell specific binding peptides by phage display is feasible. Moreover, the peptide-conjugated quantum dots may be applicable for embryonic stem cell study and utilization.

| S-EPMC2919412 | biostudies-literature

Transcriptome Profile of Quantum Dots on Saccharomyces cerevisiae

Project description:Transcriptome Profile of CdSe/ZnS and InP/ZnS Quantum Dots on Saccharomyces cerevisiae

2021-01-30 | GSE165805 | GEO