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Methods in Molecular Biology (Clifton,... 2015The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been...
The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.
Topics: Biotin; Biotinylation; Carbon-Nitrogen Ligases; Chromatography, Affinity; Escherichia coli; Escherichia coli Proteins; Glutathione Transferase; Polymerase Chain Reaction; Protein Binding; Protein Engineering; Recombinant Fusion Proteins; Repressor Proteins; Streptavidin
PubMed: 25560075
DOI: 10.1007/978-1-4939-2272-7_12 -
Genomics, Proteomics & Bioinformatics Feb 2022Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein...
Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
Topics: Ascorbate Peroxidases; Biotin; Biotinylation; Histone Code; Histones; Nucleosomes; Proteome; Staphylococcal Protein A; Streptavidin
PubMed: 34555496
DOI: 10.1016/j.gpb.2021.09.003 -
Biochemical and Biophysical Research... Dec 2017Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to...
Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.
Topics: Animals; Biotin; Biotinylation; Blotting, Western; Carbon-Nitrogen Ligases; Dogs; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Hot Temperature; Immunoprecipitation; Madin Darby Canine Kidney Cells; Proteins; Recombinant Proteins; Repressor Proteins; Solubility; Streptavidin
PubMed: 28986262
DOI: 10.1016/j.bbrc.2017.09.168 -
Molecular Cancer Sep 2022Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide...
BACKGROUND
Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma.
METHODS
The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways.
RESULTS
The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects.
CONCLUSIONS
This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.
Topics: Aquaporin 2; Carcinoma, Renal Cell; Cell Line, Tumor; Epiregulin; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Molecular Docking Simulation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Streptavidin
PubMed: 36117171
DOI: 10.1186/s12943-022-01654-1 -
Annual Review of Plant Biology May 2023Proteins are workhorses in the cell; they form stable and more often dynamic, transient protein-protein interactions, assemblies, and networks and have an intimate... (Review)
Review
Proteins are workhorses in the cell; they form stable and more often dynamic, transient protein-protein interactions, assemblies, and networks and have an intimate interplay with DNA and RNA. These network interactions underlie fundamental biological processes and play essential roles in cellular function. The proximity-dependent biotinylation labeling approach combined with mass spectrometry (PL-MS) has recently emerged as a powerful technique to dissect the complex cellular network at the molecular level. In PL-MS, by fusing a genetically encoded proximity-labeling (PL) enzyme to a protein or a localization signal peptide, the enzyme is targeted to a protein complex of interest or to an organelle, allowing labeling of proximity proteins within a zoom radius. These biotinylated proteins can then be captured by streptavidin beads and identified and quantified by mass spectrometry. Recently engineered PL enzymes such as TurboID have a much-improved enzymatic activity, enabling spatiotemporal mapping with a dramatically increased signal-to-noise ratio. PL-MS has revolutionized the way we perform proteomics by overcoming several hurdles imposed by traditional technology, such as biochemical fractionation and affinity purification mass spectrometry. In this review, we focus on biotin ligase-based PL-MS applications that have been, or are likely to be, adopted by the plant field. We discuss the experimental designs and review the different choices for engineered biotin ligases, enrichment, and quantification strategies. Lastly, we review the validation and discuss future perspectives.
Topics: Biotin; Organelles; Proteins; Streptavidin; Plants
PubMed: 36854476
DOI: 10.1146/annurev-arplant-070522-052132 -
The Journal of Cell Biology Sep 2021Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been...
Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.
Topics: Animals; Antibodies; Biotin; Cell Line, Tumor; Fluorescent Dyes; HEK293 Cells; HeLa Cells; Humans; Mice; Microscopy, Fluorescence; Microtubules; Mouse Embryonic Stem Cells; Osteoblasts; Staining and Labeling; Streptavidin; Succinimides
PubMed: 34228783
DOI: 10.1083/jcb.202105067 -
Chemical Society Reviews May 2017Biotin/(strept)avidin self-assembly is a powerful platform for nanoscale fabrication and capture with many different applications in science, medicine, and... (Review)
Review
Biotin/(strept)avidin self-assembly is a powerful platform for nanoscale fabrication and capture with many different applications in science, medicine, and nanotechnology. However, biotin/(strept)avidin self-assembly has several well-recognized drawbacks that limit performance in certain technical areas and there is a need for synthetic mimics that can either become superior replacements or operational partners with bio-orthogonal recognition properties. The goal of this tutorial review is to describe the recent progress in making high affinity synthetic association partners that operate in water or biological media. The review starts with a background summary of biotin/(strept)avidin self-assembly and the current design rules for creating synthetic mimics. A series of case studies are presented that describe recent success using synthetic derivatives of cyclodextrins, cucurbiturils, and various organic cyclophanes such as calixarenes, deep cavitands, pillararenes, and tetralactams. In some cases, two complementary partners associate to produce a nanoscale complex and in other cases a ditopic host molecule is used to link two partners. The article concludes with a short discussion of future directions and likely challenges.
Topics: Avidin; Biotin; Calixarenes; Cyclodextrins; Humans; Macrocyclic Compounds; Streptavidin
PubMed: 28191579
DOI: 10.1039/c7cs00011a -
Molecular Systems Biology May 2020Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived...
Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.
Topics: Arginine; Biotinylation; Chromatin Immunoprecipitation; HeLa Cells; Humans; Lysine; Mass Spectrometry; Membrane Proteins; Metalloendopeptidases; Neoplasm Proteins; Polycomb Repressive Complex 2; Proteins; Proteolysis; Proteomics; Streptavidin; Transcription Factors; Trypsin
PubMed: 32400114
DOI: 10.15252/msb.20199370 -
Toxins Feb 2023Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways... (Review)
Review
Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.
Topics: Animals; Saporins; Immunotoxins; Streptavidin; Ribosome Inactivating Proteins, Type 1; Cell Death; Plant Proteins; N-Glycosyl Hydrolases
PubMed: 36977072
DOI: 10.3390/toxins15030181 -
Proceedings of the Japan Academy.... 2019In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes... (Review)
Review
In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes radical cure very difficult. Precision medicine against advanced cancer is hampered by the lack of systems aimed at multiple target molecules within multiple loci. Here, we report the development of a versatile diagnostic and therapeutic system for advanced cancer, named the Cupid and Psyche system. Based on the strong non-covalent interaction of streptavidin and biotin, a low immunogenic mutated streptavidin, Cupid, and a modified artificial biotin, Psyche, have been designed. Cupid can be fused with various single-chain variable fragment antibodies and forms tetramer to recognize cancer cells precisely. Psyche can be conjugated to a wide range of diagnostic and therapeutic agents against malignant cells. The Cupid and Psyche system can be used in pre-targeting therapy as well as photo-immunotherapy effectively in animal models supporting the concept of a system for precision medicine for multiple targets within multiple loci.
Topics: Animals; Antineoplastic Agents; Biotin; Cell Line, Tumor; Drug Delivery Systems; Humans; Immunotherapy; Neoplasms; Precision Medicine; Single-Chain Antibodies; Streptavidin
PubMed: 31827018
DOI: 10.2183/pjab.95.041