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Autophagy supports cell survival under different stress conditions, where ATG8-family proteins are required for autophagosome biogenesis/maturation and selective autophagy. Here, we present a protocol for studying ATG8-family protein phosphorylation using Phos-tag gel, a modified SDS-PAGE system, when the related phosphorylation site information and/or specific phospho-antibody are unavailable. We describe steps for generating GST-ATG8 proteins in bacteria, purifying S protein-Flag-SBP protein (SFB)-tagged kinasefrom cells, preparing gel, and an in vitro kinase assay. We then detail procedures for western blotting and image processing. For complete details on the use and execution of this protocol, please refer to Seo et al.1.

Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1.

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells. [ABSTRACT FROM AUTHOR]

Chemokine receptors, a subfamily of G-protein-coupled receptors (GPCRs), are responsible for cell migration during physiological processes as well as in diseases like inflammation and cancers. Here, we present a protocol for solubilizing, purifying, and reconstituting complexes of chemokine receptors with their ligands in nanodiscs, soluble lipid bilayers that mimic the native environment of membrane receptors. The protocol yields chemokine receptor complexes with sufficient purity and yield for structural and biophysical studies and should be applicable to other GPCRs.

Summary: The tumor suppressor breast cancer 1 (BRCA1) complexed with BRCA1-associated RING domain 1 (BARD1), a RING-type E3 ligase, facilitates the attachment of ubiquitin onto the substrate protein. Here, we present a protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation. We describe steps for isolating 147 bp Widom 601 DNA and assembling nucleosome core particles (NCPs). We then detail procedures for the in vitro ubiquitylation of nucleosome histone H2A by BRCA1-BARD1 and its variants.For complete details on the use and execution of this protocol, please refer to Wang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The histone lysine methyltransferase SUV420H1 preferentially targets the H2A.Z-containing nucleosome core particle (H2A.Z-NCP) and catalyzes the H4K20me2 modification at replication origins. Here, we present a protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination. We describe steps for the installation of S-ethyl-cysteine (Ecx), nucleosome and complex preparation, and performing the cryoelectron microscopy (cryo-EM) sample check. This protocol substitutes lysine 20 in histone H4 with S-ethyl-cysteine (H4K20Ecx), which enhances the stability of the interaction between SUV420H1 and nucleosomes.For complete details on the use and execution of this protocol, please refer to Huang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present an optimized acyl-PEGyl exchange gel shift (APEGS) assay to monitor palmitoylation of high-molecular-weight proteins from primary neuronal cultures. We describe steps for culturing cortical neurons from rat embryos and expressing proteins of interest. We then detail procedures for employing a fatty acyl exchange technique wherein hydroxylamine is used to cleave palmitic acid from the palmitoyl-thioester bond, exposing cysteine residues that are subsequently labeled with methoxy polyethylene glycol maleimide (mPEG-MAL-10k).For complete details on the use and execution of this protocol, please refer to Yucel et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Fragment-based drug design plays an important role in drug discovery. Protein-observed NMR experiments with isotopically labeled samples are used to probe target-ligand interactions and map the ligand-binding sites. Here, we present a protocol to perform fragment screening using NMR spectroscopy. We describe steps for producing 15N-labeled Kirsten rat sarcoma viral oncogene homolog (KRAS) G12D protein, fragment screening using 1H-15N-heteronuclear single quantum coherence (HSQC) experiment, fragment deconvolution, determining binding affinities, and mapping the fragment-binding site. This protocol provides a strategy in fragment screening. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells.For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM).For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: S-acylation of proteins allows their association with membranes. Here, we present a protocol for establishing a platform for membrane affinity evaluation of S-acylated proteins in vitro. We describe steps for preparing lipid-maleimide compounds, mCherry-p62 recombinant proteins, and total cellular membranes. We then detail procedures for synthesizing protein-lipid conjugates using lipid-maleimide compounds and recombinant proteins and evaluating the membrane affinity of protein-lipid conjugates.For complete details on the use and execution of this protocol, please refer to Huang Xue et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer.For complete details on the use and execution of this protocol, please refer to Lin et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Activating transcription factor 4 (ATF4) is a key player in the integrated stress response, whose expression is subject to tight translational control. Studying its stress-provoked induction, accompanied by the general translational shutdown, is intricate because the expression of reference genes declines rapidly, and finding appropriate normalization controls is challenging. We present a protocol for human hemagglutinin-tagged ATF4 (hATF4-HA) detection and high-throughput quantification in non-stress versus stress conditions using automated and quantitative western blotting. We describe steps for seeding cells, transfecting plasmids, thapsigargin treatment, sample preparation, and target protein detection.For complete details on the use and execution of this protocol, please refer to Smirnova et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The ribosome-associated protein quality control (RQC) core factor nuclear export mediator factor (NEMF) appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CESs compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CESs remains largely unidentified. Here, we present a protocol for identifying and characterizing NEMF-mediated C-terminal modifications on mitochondrial NCs (mitoNCs) via tandem mass spectrometry (MS/MS) analysis. We describe strategies aimed at constructing a customized MS/MS spectra database for unknown CESs and detail the steps for CES-modified sample preparation.For complete details on the use and execution of this protocol, please refer to Lv et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Engineered ascorbate peroxidase, APEX2, is widely applied for the identification of intracellular molecule-molecule interaction analyses. Here, we present a protocol for identifying interactors of RNA-binding proteins (RBPs) in living HeLa cells using the APEX2 fusion construct. We describe steps for generation of RBP-APEX2, proximity biotin labeling, and preparation of labeled molecules for mass spectrometry analysis. This protocol may be applicable to other cell cultures and RBPs of interest.For complete details on the use and execution of this protocol, please refer to Uozumi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Isolation of antigen-specific plasma B cells had been challenging until the recent arrival of the Beacon platform. Leveraging light-sorting technology, Beacon can perform high-throughput screening of plasma B cells on a chip to sort single cells with the desired antigen specificity. Here, we present a protocol for isolating antigen-specific plasma B cells from immunized mice using Beacon, sequencing the encoded B cell receptors (BCRs), and cloning and expressing the resulting antibodies. This protocol can easily be extended to human samples. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Liquid-liquid phase separation (LLPS) of scaffold proteins has often been proposed to drive the biogenesis of membraneless cellular compartments. Here, we present a protocol to link in vitro LLPS propensity to localization in vivo. We describe steps for examining LLPS in vitro in the presence of crowding agents or cytomimetic media. We complement our in vitro studies with recombinant proteins with experiments of protein electroporation into mitotic HeLa cells. In addition, we discuss steps to assess protein localization and delivery levels.For complete details on the use and execution of this protocol, please refer to Hedtfeld et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors.For complete details on the use and execution of this protocol, please refer to Pham et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Proteins congregate into complexes to perform diverse cellular functions. Protein complexes are remodeled by protein-coding mutations or cellular signaling changes, driving phenotypic outcomes in health and disease. We present an affinity purification-mass spectrometry (AP-MS) proteomics protocol to express affinity-tagged “bait” proteins in mammalian cells, identify and quantify purified protein interactors, and visualize differential protein-protein interaction networks between pairwise conditions. Our protocol possesses general applicability to various cell types and biological areas.For complete details on the use and execution of this protocol, please refer to Bouhaddou et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Cerebral cortex biopsies enable the investigation of native developing and mature human brain tissue. Here, we present a protocol to process human cortical biopsies from the surgical theater to the laboratory. We describe steps for the preparation of viable acute slices for patch-clamp recording using dedicated chemical solutions for transport and sectioning. We then explain procedures for tissue fixation and post hoc immunostaining to correlate physiological properties to morphological features and protein detection.For complete details on the use and execution of this protocol, please refer to Libé-Philippot et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Iontophoresis enables the non-invasive transdermal delivery of moderately-sized proteins and the needle-free cutaneous delivery of antibodies. However, simple descriptors of protein characteristics cannot accurately predict the feasibility of iontophoretic transport. This study investigated the cathodal and anodal iontophoretic transport of the negatively charged M7D12H nanobody and a series of negatively charged variants with single amino acid substitutions. Surprisingly, M7D12H and its variants were only delivered transdermally by anodal iontophoresis. In contrast, transdermal permeation after cathodal iontophoresis and passive diffusion was

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.

Summary: Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Cellular protein homeostasis is maintained by the disposal of aggregated misfolded proteins. Here, we present a protocol for investigating the involvement of the proteins of interest in misfolded protein degradation via Agrobacterium-mediated transient expression in Nicotiana benthamiana. We describe in detail the steps of misfolded protein design, transient protein expression in N. benthamiana, subsequent total protein extraction, and quantification of misfolded proteins through western blotting. This generalizable system can be used for misfolded proteins derived from various plants or microbes.For complete details on the use and execution of this protocol, please refer to Ai et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Flavonoids are naturally occurring metabolites of plants that can inhibit the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), which is required for viral replication. Here, we present a protocol to identify flavonoid antagonists of the SARS-CoV-2 Mpro. We describe steps for the expression and purification of Mpro and a kinetic enzymatic assay for Mpro activity using a dequenching fluorescence resonance energy transfer peptide substrate. We then detail procedures for using this enzymatic assay to test flavonoid antagonism and reversible inhibition.For complete details on the use and execution of this protocol, please refer to Lin et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The minor phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is crucial for neurotransmission and has been implicated in Parkinson’s disease. Here, we present a staining protocol for the analysis of activity-dependent changes of PI(4,5)P2 at synapses. We describe steps for stimulating and fixing murine hippocampal neurons, staining with probes for PI(4,5)P2 and a synaptic marker, and analysis by high-resolution microscopy. Our approach gives insights into local PI(4,5)P2 synthesis and turnover at synapses and can be extended to phosphoinositide lipids other than PI(4,5)P2.For complete details on the use and execution of this protocol, please refer to Bolz et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor that can be targeted for inducing potent adjuvant effects. Mincle can recognize trehalose dimycolate and related glycolipids. Here, we present a protocol to identify the ligand binding mode of Mincle. We describe steps for preparing labeled Mincle ectodomain, data acquisition, and analysis of nuclear magnetic resonance experiments using non-detergent sulfobetaine-195. This protocol can be applied to other protein-ligand interactions that have aggregation problems for complex formation.For complete details on the use and execution of this protocol, please refer to Furukawa et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: 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.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers.For complete details on the use and execution of this protocol, please refer to Tang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM.For complete details on the use and execution of this protocol, please refer to Neville et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The growing prevalence of hyperuricemia necessitates the urgent development of more potent treatments. This study aimed to develop, optimize, and evaluate the safety and efficacy of porcine–human recombinant uricase (PHRU) both in vitro and in vivo. The study employed gene editing of PHRU through site-directed mutagenesis, with recombinant proteins expressed in vitro utilizing Escherichia coli. The polyethylene glycol (PEG) approach was employed to augment uricase stability and diminish immunogenicity. The pharmacokinetics and pharmacodynamics of PHRU were tested in vitro and in Sprague Dawley rats. Successful expression of the fusion protein in E. coli and the development of the PEGylated drug were achieved. In vitro experiments confirmed the efficacy of PEG-PHRU in degrading uric acid, with PEGylation not markedly affecting the biological activity of PHRU. Animal studies revealed that PEG-PHRU significantly lowered plasma uric acid levels and mitigated hyperuricemia-induced renal damage in rats. Both drug metabolism and pharmacokinetics exhibited favorable characteristics without observable adverse effects in experimental animals. This novel fusion protein shows the potential for ameliorating hyperuricemia and related renal complications, highlighting it as a promising drug candidate with substantial market applications.

Summary: Autophagy supports cell survival under different stress conditions, where ATG8-family proteins are required for autophagosome biogenesis/maturation and selective autophagy. Here, we present a protocol for studying ATG8-family protein phosphorylation using Phos-tag gel, a modified SDS-PAGE system, when the related phosphorylation site information and/or specific phospho-antibody are unavailable. We describe steps for generating GST-ATG8 proteins in bacteria, purifying S protein-Flag-SBP protein (SFB)-tagged kinasefrom cells, preparing gel, and an in vitro kinase assay. We then detail procedures for western blotting and image processing.For complete details on the use and execution of this protocol, please refer to Seo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Anuran saxiphilins (Sxphs) are “toxin sponge” proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins.For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. Results In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72–97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. Conclusion Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

Summary: G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners.For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action.For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a pipeline for the characterization of synaptic structural plasticity in mouse spinal dorsal horn (SDH) neurons. We describe steps for the intra-SDH microinjection of the EGFP virus to sparsely label L4 SDH neurons without laminectomy, wide dynamic range neuron imaging, dendritic spine morphometric analysis, and F-actin to G-actin ratio measurement. This protocol can be applied to investigate the synaptic structural plasticity mechanisms in the SDH as well as in the brain.For complete details on the use and execution of this protocol, please refer to Li et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Proteasomes are heterogeneous in forms and functions, but how the equilibrium among the 20S, 26S, and 30S proteasomes is achieved and altered is elusive. Here, we present a protocol for purifying and characterizing proteasome species. We describe steps for generating stable cell lines; affinity purifying the proteasome species; and characterizing them through native PAGE, activity assay, size-exclusion chromatography, and mass spectrometry. These standardized methods may contribute to biochemical studies of cellular proteasomes under both physiological and pathological conditions.For complete details on the use and execution of this protocol, please refer to Choi et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Dynamic macromolecular complexes containing a large number of components are often difficult to study using conventional approaches, such as immunoblotting. Here, we present a protocol for the analysis of macromolecular complexes in near-native conditions using a flexible setup to suit different cellular targets. We describe analysis of human mitochondrial ribosome, composed of 82 proteins, in a standardized way using density gradient ultracentrifugation coupled to quantitative mass spectrometry and subsequent analysis of the generated data (ComPrAn).For complete details on the use and execution of this protocol, please refer to Páleníková et al.1 and Rebelo-Guiomar et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We then detail procedures for verification of LtCas12a with cis- and trans-cleavage activities.For complete details on the use and execution of this protocol, please refer to Chen et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types.For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background: Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. Results: In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72–97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. Conclusion: Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation. [ABSTRACT FROM AUTHOR]

Objective: The prokaryotic expression vector pGEX-4T-3/Myo5a was prepared, the fusion protein was expressed and purified, and polyclonal antibody was prepared. Methods: One small fragment of Myo5a was amplified by PCR. After BamHⅠ and XhoⅠdouble digestion, the fragments were inserted into prokaryotic expression vector pGEX-4T-3 to construct one prokaryotic expression vectors pGEX-4T-3/Myo5a. After correct identification, BL21 prokaryotic expression bacteria were transferred to IPTG induction. The recombinant protein pGEX-4T-3/Myo5a was purified and the recombinant protein was identified by SDS-PAGE electrophoresis and Western blot. The rabbit was immunized with fusion protein, whose antiserum was prepared and its titer and specificity were determined. Results: After identification, the prokaryotic expression vector pGEX-4T-3/Myo5a was prepared correctly, and the relative molecular weight of the expressed recombinant protein was about 30 kD. After immunizing rabbits, the antiserum titer was 1∶ 128 000, and the specificity was proved by Western blot and indirect immunofluorescence. Conclusion: The PGEX-4T-3/Myo5a prokaryotic expression vector is successfully prepared, the fusion protein had high immune reactivity, and its polyclonal antibody is obtain, which paved the way for further exploration of the biological significance of Myo5a. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9’s main element Cas9 protein is generally expressed by Escherichia coli, but in the process of expression and purification, Cas9 protein is prone to problems such as the formation of insoluble inclusion bodies, high endotoxin content, incorrect protein folding due to too large protein molecules, and low yield. This study aimed to achieve efficient soluble expression of Streptococcus pyogenes Cas9 (SpCas9) protein in E. coli for the purpose of promoting its application and popularizing gene editing technology. The solubility-enhancing tag GB1 was applied to improve the expression level and solubility of Cas9 protein, and a multiple promoter strategy was used to further improve the expression level of Cas9 protein. As a result, the expression of Cas9 protein was increased by 3.52 times. In vitro enzymatic digestion analysis showed that the functional activity of Cas9 protein was not affected by fusion with GB1. Furthermore, a ribonucleoprotein (RNP) complex was assembled and transformed into the host Aspergillus niger, so that the pyrG gene was successfully destroyed.

Iontophoresis enables the non-invasive transdermal delivery of moderately-sized proteins and the needle-free cutaneous delivery of antibodies. However, simple descriptors of protein characteristics cannot accurately predict the feasibility of iontophoretic transport. This study investigated the cathodal and anodal iontophoretic transport of the negatively charged M7D12H nanobody and a series of negatively charged variants with single amino acid substitutions. Surprisingly, M7D12H and its variants were only delivered transdermally by anodal iontophoresis. In contrast, transdermal permeation after cathodal iontophoresis and passive diffusion was WT (wild type), and the R54E and K65E variants, was statistically superior to that after cathodal iontophoresis (6.07 ± 2.11, 9.22 ± 0.80, and 14.45 ± 3.45 μg/cm2, versus 1.12 ± 0.30, 0.72 ± 0.27, and 0.46 ± 0.07 µg/cm2, respectively). This was not the case for S102E, where cutaneous deposition after anodal and cathodal iontophoresis was 11.89 ± 0.87 and 8.33 ± 2.62 µg/cm2, respectively; thus, a single amino acid substitution appeared to be sufficient to impact the iontophoretic transport of a 17.5 kDa protein. Visualization studies using immunofluorescent labeling showed that skin transport of M7D12HWT was achieved via the intercellular and follicular routes.

Summary: Biosynthetic gene clusters of natural products often harbor genes of unknown function, which are difficult to characterize. Here, we present a protocol for the functional analysis in vitro and in vivo of these biosynthetic genes by heterologous expression in E. coli. We describe steps for the expression of genes of interest in an established E. coli strain optimized to heterologously express natural products. We then detail the expression of a His-tagged gene to deduce the specific function of the protein.For complete details on the use and execution of this protocol, please refer to Böhringer et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a detailed protocol to study the role of a human nuclear m6A RNA reader, YTHDC1, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins.For complete details on the use and execution of this protocol, please refer to Timcheva et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: In vitro validation of therapeutic and recombinant proteins expressed from transgenic chickens is limited by the co-culture of fibroblasts. Here, we present a protocol for isolating pure epithelial cells derived from the magnum tubular glands of the chicken oviduct. We describe steps for preparing solutions and buffers, tissue collection, processing, dissociation, and Percoll density centrifugation to separate the epithelial cells from co-isolated fibroblasts. We then detail procedures for expressing a recombinant IgG antibody in the Percoll-derived epithelial cell line. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.