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Biochemistry and Molecular Biology... Jul 2021Western blot (WB) or immunoblot is a workhorse method. It is commonly used by biologists for study of different aspects of protein biomolecules. In addition, it has been... (Review)
Review
Western blot (WB) or immunoblot is a workhorse method. It is commonly used by biologists for study of different aspects of protein biomolecules. In addition, it has been widely used in disease diagnosis. Despite some limitations such as long time, different applications of WB have not been limited. In the present review, we have summarized scientific and clinical applications of WB. In addition, we described some new generation of WB techniques.
Topics: Blotting, Western; Humans; Proteins; Translational Research, Biomedical
PubMed: 33847452
DOI: 10.1002/bmb.21516 -
Protein Expression and Purification Aug 2020Antibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When... (Review)
Review
Antibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or post-translational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.
Topics: Animals; Gene Expression; Humans; Protein Folding; Protein Stability; Recombinant Proteins; Single-Domain Antibodies
PubMed: 32289357
DOI: 10.1016/j.pep.2020.105645 -
Methods in Molecular Biology (Clifton,... 2020MxB/Mx2 is an interferon-induced dynamin-like GTPase, which restricts a number of life-threatening viruses. Because of its N-terminal region, predicted to be...
MxB/Mx2 is an interferon-induced dynamin-like GTPase, which restricts a number of life-threatening viruses. Because of its N-terminal region, predicted to be intrinsically disordered, and its propensity to self-oligomerize, purification of the full-length protein has not been successful in conventional E. coli expression systems. In this chapter, we describe an expression and purification procedure to obtain pure full-length wild-type MxB from suspension-adapted mammalian cells. We further describe how to characterize its GTPase activity and oligomerization function.
Topics: Animals; Chromatography, Affinity; Chromatography, Gel; Enzyme Activation; GTP Phosphohydrolases; Gene Expression; HEK293 Cells; Humans; Myxovirus Resistance Proteins; Plasmids; Recombinant Fusion Proteins; Transfection
PubMed: 32529363
DOI: 10.1007/978-1-0716-0676-6_5 -
Biochemical Society Transactions Jun 2021In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological... (Review)
Review
In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein-lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein-protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.
Topics: Cryoelectron Microscopy; Lipid Bilayers; Maleates; Membrane Lipids; Membrane Proteins; Polystyrenes; Protein Binding; Protein Conformation; Protein Stability
PubMed: 34110372
DOI: 10.1042/BST20201067 -
Journal of Neurochemistry Apr 2020Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their...
Disentangling aggregation-prone proteins: a new method for isolating α-synuclein species: An Editorial Highlight for "A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: Towards improving...
Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson's disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases.
Topics: Centrifugation; Filtration; Humans; Parkinson Disease; Protein Aggregation, Pathological; Reproducibility of Results; alpha-Synuclein
PubMed: 32037541
DOI: 10.1111/jnc.14973 -
Marine Drugs Aug 2021Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich... (Review)
Review
Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich composition in fiber, minerals, and proteins, has pointed them as a useful source of these components. Seaweed proteins are nutritionally valuable and include several specific enzymes, glycoproteins, cell wall-attached proteins, phycobiliproteins, lectins, or peptides. Extraction of seaweed proteins requires the application of disruptive methods due to the heterogeneous cell wall composition of each macroalgae group. Hence, non-protein molecules like phenolics or polysaccharides may also be co-extracted, affecting the extraction yield. Therefore, depending on the macroalgae and target protein characteristics, the sample pretreatment, extraction and purification techniques must be carefully chosen. Traditional methods like solid-liquid or enzyme-assisted extraction (SLE or EAE) have proven successful. However, alternative techniques as ultrasound- or microwave-assisted extraction (UAE or MAE) can be more efficient. To obtain protein hydrolysates, these proteins are subjected to hydrolyzation reactions, whether with proteases or physical or chemical treatments that disrupt the proteins native folding. These hydrolysates and derived peptides are accounted for bioactive properties, like antioxidant, anti-inflammatory, antimicrobial, or antihypertensive activities, which can be applied to different sectors. In this work, current methods and challenges for protein extraction and purification from seaweeds are addressed, focusing on their potential industrial applications in the food, cosmetic, and pharmaceutical industries.
Topics: Chemical Fractionation; Peptides; Phenols; Polysaccharides; Protein Hydrolysates; Seaweed
PubMed: 34564162
DOI: 10.3390/md19090500 -
Biochimica Et Biophysica Acta. Gene... Feb 2021Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism... (Review)
Review
Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family. It is now appreciated that protein acetylation occurs on thousands of proteins and can reversibly regulate the function of many cellular processes. In this review, we provide an overview of our fundamental understanding of catalysis, regulation of activity and substrate selection, and inhibitor development for this archetypal acetyltransferase.
Topics: Acetyl Coenzyme A; Acetylation; Biocatalysis; Crystallography; Drug Development; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Histone Acetyltransferases; Histones; Lysine; Models, Molecular; Multienzyme Complexes; Protein Domains; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Structure-Activity Relationship; Substrate Specificity; Transcriptional Activation; p300-CBP Transcription Factors
PubMed: 32841743
DOI: 10.1016/j.bbagrm.2020.194627 -
Current Opinion in Immunology Oct 2020Flaviviruses are a group of important emerging and re-emerging human pathogens that cause worldwide epidemics with thousands of deaths annually. Flaviviruses are small,... (Review)
Review
Flaviviruses are a group of important emerging and re-emerging human pathogens that cause worldwide epidemics with thousands of deaths annually. Flaviviruses are small, enveloped, positive-sense, single-stranded RNA viruses that are obligate intracellular pathogens, relying heavily on host cell machinery for productive replication. Proteomic approaches have become an increasingly powerful tool to investigate the mechanisms by which viruses interact with host proteins and manipulate cellular processes to promote infection. Here, we review recent advances in employing quantitative proteomics techniques to improve our understanding of the complex interplay between flaviviruses and host cells. We describe new findings on our understanding of how flaviviruses impact protein-protein interactions, protein-RNA interactions, protein abundance, and post-translational modifications to modulate viral infection.
Topics: Animals; Flavivirus; Host-Pathogen Interactions; Humans; Protein Binding; Proteins; Proteomics; Virus Replication
PubMed: 32682290
DOI: 10.1016/j.coi.2020.06.002 -
The FEBS Journal Oct 2021Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures... (Review)
Review
Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures is essential for understanding their functions, and this information can also be exploited for structure-aided drug discovery efforts. In this regard, X-ray crystallography has been the most widely used technique for determining the high-resolution 3D structures of membrane proteins. However, the success of this technique is dependent on efficient protein extraction, solubilization, stabilization, and generating diffracting crystals. Each of these steps can impose great challenges for membrane protein crystallographers. In this review, the process of generating membrane protein crystals from protein extraction and solubilization to structure determination is discussed. In addition, the current methods for precrystallization screening and a few strategies to increase the chance of crystallizing challenging membrane proteins are introduced.
Topics: Animals; Crystallization; Crystallography, X-Ray; Humans; Membrane Proteins; Protein Conformation
PubMed: 33340246
DOI: 10.1111/febs.15676 -
Protein Expression and Purification Mar 2020Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel... (Review)
Review
Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.
Topics: Animals; Cell Line; Cloning, Molecular; Drosophila melanogaster; Drug Delivery Systems; Drug Design; Gene Expression; Maleates; Polystyrenes; Receptors, G-Protein-Coupled; Recombinant Proteins; Solubility
PubMed: 31678667
DOI: 10.1016/j.pep.2019.105524