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Immunity Aug 2023Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our...
Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.
Topics: Antibodies, Monoclonal; Antibodies, Viral; Influenza A Virus, H1N1 Subtype; Neuraminidase; Humans; Immunoglobulin Fab Fragments; Cryoelectron Microscopy; Epitopes; Mice, Inbred BALB C; Animals; Mice; Influenza, Human; Disease Models, Animal
PubMed: 37506693
DOI: 10.1016/j.immuni.2023.07.004 -
Nature Protocols Jan 2024The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we... (Review)
Review
The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENP. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.
Topics: Humans; Proteins; Peptides; Proteolysis; Recombinant Fusion Proteins; Chromatography, Affinity
PubMed: 37974029
DOI: 10.1038/s41596-023-00904-w -
Nature Protocols Sep 2023Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many... (Review)
Review
Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.
Topics: Animals; Caenorhabditis elegans; Animals, Genetically Modified; Membrane Proteins; Chromatography, Gel; Cell Line; Caenorhabditis elegans Proteins
PubMed: 37495753
DOI: 10.1038/s41596-023-00852-5 -
Protein and Peptide Letters 2024DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of...
BACKGROUND
DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation.
OBJECTIVES
The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation.
METHODS
DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography.
RESULTS
The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18°C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity.
CONCLUSION
A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3.
Topics: DEAD-box RNA Helicases; Escherichia coli; Chromatography, Affinity; Humans; Recombinant Fusion Proteins; Cloning, Molecular; Gene Expression
PubMed: 38303525
DOI: 10.2174/0109298665285625231222075700 -
Protein Science : a Publication of the... Jun 2024While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial...
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
Topics: Histidine; Polyphosphates; Nitrilotriacetic Acid; Recombinant Proteins; Humans; Proteins
PubMed: 38747394
DOI: 10.1002/pro.5021 -
Protein Expression and Purification Sep 2024Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles...
Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl β-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.
Topics: Galectin 3; Escherichia coli; Humans; Isopropyl Thiogalactoside; Gene Expression; Galectins; Recombinant Proteins; Blood Proteins
PubMed: 38801985
DOI: 10.1016/j.pep.2024.106516 -
STAR Protocols Jun 2024The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein,...
The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives. For complete details on the use and execution of this protocol, please refer to Kim et al..
Topics: Crystallization; Dinitrobenzenes; Crystallography, X-Ray; Transcription Factors; Bacterial Proteins
PubMed: 38573865
DOI: 10.1016/j.xpro.2024.102999 -
The Analyst Apr 2024Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology,... (Review)
Review
Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.
Topics: Humans; Point-of-Care Testing; Viruses; Virus Diseases; Antigens, Viral; Antibodies, Viral; Biosensing Techniques; Point-of-Care Systems; RNA, Viral
PubMed: 38630498
DOI: 10.1039/d4an00238e -
Current Protocols Jun 2024U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes....
U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.
Topics: Escherichia coli; Humans; Ribonucleoprotein, U1 Small Nuclear; Phosphorylation; Recombinant Proteins; Gene Expression; Protein Domains
PubMed: 38896106
DOI: 10.1002/cpz1.1059 -
Current Protocols Jan 2024Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell...
Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.
Topics: Hydrogels; Phosphoproteins; Endopeptidases; Alginates; Biocompatible Materials; Collagen
PubMed: 38206582
DOI: 10.1002/cpz1.966