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Methods in Molecular Biology (Clifton,... 2017Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidence shows that protein-protein interactions are crucial...
Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidence shows that protein-protein interactions are crucial in many biological processes in living cells. Genetic (such as yeast two-hybrid, Y2H) and biochemical (such as co-immunoprecipitation, co-IP) methods are the methods commonly used at the beginning of a study to identify the interacting proteins. Immunoprecipitation (IP), a method using a target protein-specific antibody in conjunction with Protein A/G affinity beads, is a powerful tool to identify molecules that interact with specific proteins. Therefore, co-IP is considered to be one of the standard methods of identifying or confirming the occurrence of protein-protein interaction events in vivo. Co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use Agrobacterium type VI secretion system (T6SS) sheath components TssB-TssC interaction as an example to describe the principle, procedure, and experimental problems of co-IP.
Topics: Agrobacterium; Bacterial Proteins; Immunoprecipitation; Multiprotein Complexes; Protein Binding; Protein Interaction Mapping; Proteins; Staphylococcal Protein A
PubMed: 28667615
DOI: 10.1007/978-1-4939-7033-9_17 -
Journal of Molecular Biology Nov 2018The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In...
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
Topics: Animals; Cell Nucleus; Chemical Fractionation; Cytoplasm; Guidelines as Topic; In Vitro Techniques; Liquid-Liquid Extraction; Peptide Termination Factors; Phase Transition; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Solid Phase Extraction
PubMed: 29944854
DOI: 10.1016/j.jmb.2018.06.038 -
Methods in Molecular Biology (Clifton,... 2017All cells contain proteases which hydrolyze the peptide bonds between amino acids in a protein backbone. Typically, proteases are prevented from nonspecific proteolysis...
All cells contain proteases which hydrolyze the peptide bonds between amino acids in a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; firstly inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographical purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing or avoiding proteolysis during protein purification and subsequent chromatography are reviewed.
Topics: Chromatography; Hydrolysis; Peptide Hydrolases; Protease Inhibitors; Proteins; Proteolysis; Recombinant Proteins
PubMed: 27730548
DOI: 10.1007/978-1-4939-6412-3_4 -
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 -
Protein Expression and Purification Dec 2015Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate...
Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin.
Topics: Bacterial Proteins; Base Sequence; Carrier Proteins; Cloning, Molecular; Cyanobacteria; Cysteine Endopeptidases; Escherichia coli; Genetic Vectors; Helicobacter pylori; Lectins; Membrane Proteins; Proteolysis; Pseudomonas; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Small Ubiquitin-Related Modifier Proteins; Solubility
PubMed: 26297996
DOI: 10.1016/j.pep.2015.08.019 -
Cold Spring Harbor Protocols Aug 2020Chromatin immunoprecipitation, commonly referred to as ChIP, is a powerful technique for the evaluation of in vivo interactions of proteins with specific regions of...
Chromatin immunoprecipitation, commonly referred to as ChIP, is a powerful technique for the evaluation of in vivo interactions of proteins with specific regions of genomic DNA. Formaldehyde is used in this technique to cross-link proteins to DNA in vivo, followed by the extraction of chromatin from cross-linked cells and tissues. Harvested chromatin is sheared and subsequently used in an immunoprecipitation incorporating antibodies specific to protein(s) of interest and thus coprecipitating and enriching the cross-linked, protein-associated DNA. The cross-linking process can be reversed, and protein-bound DNA fragments of optimal length ranging from 200 to 1000 base pairs (bp) can subsequently be purified and measured or sequenced by numerous analytical methods. In this protocol, two different fixation methods are described in detail. The first involves the standard fixation of cells and tissue by formaldehyde if the target antigen is highly abundant. The dual cross-linking procedure presented at the end includes an additional preformaldehyde cross-linking step and can be especially useful when the target protein is in low abundance or if it is indirectly associated with chromatin DNA through another protein.
Topics: Antibodies; Chromatin; Chromatin Immunoprecipitation; Cross-Linking Reagents; DNA; Magnetic Phenomena; Microspheres
PubMed: 32747583
DOI: 10.1101/pdb.prot098665 -
Methods in Molecular Biology (Clifton,... 2019Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular...
Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.
Topics: Fluorescent Dyes; Liposomes; Membrane Fusion; Phospholipids; Protein Binding; Proteolipids; Recombinant Proteins; SNARE Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30317514
DOI: 10.1007/978-1-4939-8760-3_20 -
Cold Spring Harbor Protocols Dec 2017This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody...
This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody bound to a solid matrix. Purified antigen(s) can be eluted by various methods, and the resultant protein target can be analyzed and/or identified by numerous assays, including the enzyme-linked immunosorbent assay (ELISA), western blotting, or mass spectrometry.
Topics: Antigens; Immunoprecipitation; Proteins
PubMed: 29196601
DOI: 10.1101/pdb.prot098640 -
Analytical Biochemistry Jul 2017Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant...
Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity. We report here a novel method to lyse E. coli, which is rapid, and results in high yield of isolated protein. Here, we have carried out intracellular expression of lysozyme domain (LD) of mycobacteriophage D29 endolysin. LD remains non-toxic until chloroform is added into the culture medium that permeabilizes bacterial cell membrane and allows the diffusion of LD to the peptidoglycan layer causing latter's degradation ensuing cell lysis. Our method efficiently lyses E. coli in short duration. As a proof-of-concept, we demonstrate large scale isolation and purification of α subunit of E. coli RNA polymerase and GFP, when they are co-expressed with LD. We believe that our method will be adopted easily in high-throughput as well as large scale protein isolation experiments.
Topics: Bacteriolysis; Chromatography, Affinity; Cloning, Molecular; DNA-Directed RNA Polymerases; Endopeptidases; Escherichia coli; Gene Expression; Recombinant Proteins
PubMed: 28431999
DOI: 10.1016/j.ab.2017.04.009 -
Nature Apr 2017Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human...
Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.
Topics: Adenosine Deaminase; Alu Elements; Animals; Cell Line; DEAD-box RNA Helicases; Evolution, Molecular; Exons; Gene Expression Regulation; Genes, Reporter; Genome, Human; HEK293 Cells; Humans; Inverted Repeat Sequences; Male; Mice; Mutagenesis; Neoplasm Proteins; Nucleic Acid Conformation; Protein Binding; Protein Biosynthesis; Protein Isoforms; RNA; RNA Editing; RNA, Circular; RNA, Double-Stranded; RNA-Binding Proteins; Transcription, Genetic
PubMed: 28355180
DOI: 10.1038/nature21715