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Summary: Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays.For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).

Locomotion through spatially confining spaces is an important in vivo migration mode. Here, we present a protocol for in situ visualization of mitochondrial reactive oxygen species and apoptosis in cancer cells during confined migration. We then detail sample preparation of confined cells for transcriptome and immunoblotting analysis by using transwell chambers. This approach allows in situ evaluation of a variety of cellular functions during confined migration and preparation of the samples of confined cells for further biochemical analysis. For complete details on the use and execution of this protocol, please refer to Cai et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application prospects, immunogenicity and nonspecific cytotoxicity of immunotoxins with toxins from bacterial or plant origin have hindered their clinical application. In this study, an anti-HER2 single-chain antibody fragment 4D5, was fused to a human-origin antitumor drug, TRAIL to produce a humanized immunotoxin in Escherichia coli BL21 (DE3). An appropriate linker between the domains was selected using the software Antheprot 5.0. The 4D5 scFv-TRAIL was obtained using a SUMO tag for solubility-enhancing and purified by Ni–NTA affinity chromatography after tag removing. The purity and yield of 4D5 scFv-TRAIL reached 92% and 11.6 mg/L in shake flasks, respectively. Additionally, MTT assays showed 4D5 scFv-TRAIL exhibited selective in vitro cytotoxicity on the breast cancer cell line MDA-MB-231 without cytotoxic effect on the normal cell FBHE. Therefore, 4D5 scFv-TRAIL is a potential humanized immunotoxin for treatment of HER2-overexpressing tumor cells. [ABSTRACT FROM AUTHOR]

RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 β-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology. [ABSTRACT FROM AUTHOR]

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli. [ABSTRACT FROM AUTHOR]

Human BiP/GRP78 is involved in the folding and assembly of proteins in the endoplasmic reticulum. The proteins for crystallization in good amount and quality are prerequisites for obtaining ideal crystals. To meet these requirements, different BiP/GRP78 constructs, competent cells, vectors, and concentrations of inducer were tested in order to obtain soluble BiP/GRP78 protein with the highest amount and best purity. The BiP–T229A–L3,4′–Smt3 fusion protein was expressed in a soluble manner and finally purified with the highest purity using size exclusion chromatography, which was suitable for further protein crystallization.

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasma extracellular vesicles (EVs) represent a potential resource for biomarkers of multiple diseases. Here, we present a protocol for obtaining EVs from human plasma using asymmetrical flow field-flow fractionation technology. We describe steps for using tandem mass tags to perform comparative proteomic studies of a large clinical cohort. We then detail targeted quantitative analysis of differential proteins based on a parallel reaction monitoring technique. For complete details on the use and execution of this protocol, please refer to Wu et al. (2020) 1 and Li et al. (2023). 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intercalated motifs or i-Motifs (iMs) are nucleic acid structures formed by cytosine-rich sequences, which may regulate cellular processes and have broad applications in nanotechnology due to their pH-dependent nature. We have developed an iM-specific nanobody (iMbody) that can recognize iM DNA structures regardless of their sequences, making it a versatile research tool for studying iMs in various contexts. Here, we provide a protocol for the bacterial expression and His-tag purification of iMbody. We then describe procedures for performing ELISA and immunostaining using iMbody., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytidine deaminases as DNA mutators play important roles in immunity and genome stability. Here, we present a high-throughput protocol for deamination of long single-stranded (ss) DNA or oligo pools containing complex sequences. We describe steps for the preparation of both enzyme (activation-induced deaminase, AID) and ssDNA substrates, the deamination reaction, uracil-friendly amplification, and data analysis. This assay can be used to determine the intrinsic mutation profile of a single antibody gene or a pool of selected regions on genomic DNA. For complete details on the use and execution of this protocol, please refer to Wang et al. (2023). 1 ., Competing Interests: Declaration of interests Shanghai Institute of Biochemistry and Cell Biology and Shanghai Jiao Tong University School of Medicine have filed a patent application based on the protocol in this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests M.M. is a founder, shareholder, and member of the Scientific Advisory Board of Pretzel Therapeutics, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polyphenol oxidases (PPOs) are important enzymes that are widely found in both prokaryotes and eukaryotes including grapes. Studies of grape PPO to date have mostly relied on enzymes extracted and purified from plants. In this work, we describe the production of the mature form of Shine Muscat grape PPO by using an Escherichia coli expression system. We have optimised the purification procedure to obtain pure and active recombinant enzymes and characterised the catalytic efficiency of the recombinant grape PPO by using ultraviolet/visible (UV/Vis) spectrophotometry. Our work provides a simple protocol of obtaining pure and active recombinant grape PPO that will enable further studies about the catalytic mechanism and inhibition of this enzyme. • Mature grape polyphenol oxidase was produced in Escherichia coli. • Purification protocol was optimised to produce active enzymes. • First report of active, mature polyphenol oxidase from a eukaryotic source produced in E. coli. [ABSTRACT FROM AUTHOR]

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes. [ABSTRACT FROM AUTHOR]

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

Here, we present a protocol for studying the cell-surface proteins in hippocampal slices after in vivo administration of sevoflurane, an inhaled general anesthetic drug, to mice. We describe steps for anesthetic delivery, hippocampal slice preparation, and cell-surface biotinylation. We then detail the isolation of surface proteins and their quantification through Western blotting. This protocol can be adapted to study changes in other surface proteins following exposure to various general anesthetic drugs. For complete details on the use and execution of this protocol, please refer to Wang et al. (2012), 1 Zurek et al. (2014), 2 and Yu et al. (2019). 3 ., Competing Interests: Declaration of interests B.A.O. serves on the Board of Trustees of the International Anesthesia Research Society (San Francisco, CA, USA) and is the co-director of the Perioperative Brain Health Centre (Toronto, ON, Canada http://www.perioperativebrainhealth.com). She is a named inventor on a Canadian patent (2,852,978) and two US patents (9,517,265 and 10, 981, 954). The patents, which are held by the University of Toronto, are for new methods to prevent and treat delirium and persistent neurocognitive deficits after anesthesia and surgery and to treat mood disorders. B.A.O. also collaborates on clinical studies that are supported by in-kind software., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bioorthogonal labeling and click chemistry techniques allow the detailed examination of cellular physiology through tagging and visualizing newly synthesized proteins. Here, we describe three methods applying bioorthogonal non-canonical amino acid tagging and fluorescent non-canonical amino acid tagging to quantify protein synthesis in microglia. We describe steps for cell seeding and labeling. We then detail microscopy, flow cytometry, and Western blotting techniques. These methods can be easily adapted for other cell types to explore cellular physiology in health and disease. For complete details on the use and execution of this protocol, please refer to Evans et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine. For complete details of this protocol, please refer to Liang et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In membrane proteins, a transmembrane region and a juxtamembrane region play important roles in its function. Here, we present a protocol for characterizing membrane protein dynamics between the juxtamembrane region of the single transmembrane protein and acidic membrane. We describe steps for solid-phase peptide synthesis, peptide purification, and labeling. We then detail reconstitution of the transmembrane peptide into lipid bilayers and its evaluation and structural analysis. For complete details on the use and execution of this protocol, please refer to Prasada Rao et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer. 1 Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity. For complete details on the use and execution of this protocol, please refer to Parkins et al. 2 , 3 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests Dr. Handel is a co-founder of Lassogen Inc and serves on the Scientific Advisory Boards of Artica, Abilita Bio, and Abalone Bio. The terms of these arrangements have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 ., Competing Interests: Declaration of interests Q.Z., A.M., and N.W. are inventors of a pending patent related to the antibody engineering technology submitted by Kyoto University to the Japan Patent Office. Q.Z. is an employee of Takeda Pharmaceutical Company Limited., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The trans -translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans -translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis ( Mtb SmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works have been conducted to prepare, characterize biophysical properties and determine three-dimensional structure for the full-length Mtb SmpB protein. In the present work, we successfully expressed and purified the His-tagged full-length Mtb SmpB protein in Escherichia coli with a yield of 26.9 mg from 1 L of Luria Bertani medium. We also obtained Mtb SmpB with a yield of 18.5 mg from 1 L of M9 minimal medium. The Mtb SmpB protein showed a single band in SDS-PAGE with a molecular weight of ∼20 kDa consistent with the measurement from MALDI-TOF-mass spectrometry. The dynamic light scattering experiment indicated that Mtb SmpB existed in a monomeric form. Moreover, both circular dichroism and nuclear magnetic resonance (NMR) experiments exhibited that Mtb SmpB was well structured, suggesting that it could be feasible to determine its solution structure by NMR spectroscopy. NMR titration experiments showed that Mtb SmpB specifically bound to tmRNA. This work lays the essential basis for further determining the solution structure and dynamics of the full-length Mtb SmpB protein. [ABSTRACT FROM AUTHOR]

Escherichia coli is an essential host for large-scale expression of heterologous polypeptides. However, further applications are limited by the formation of potential protein aggregates. In this work, we developed a novel on-column tag removal and purification system based on Fh8 hydrophobic interaction chromatography purification and ΔI-CM self-cleavage to obtain soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We evaluated several methods to improve TRAIL solubility and finally demonstrated that the Fh8 tag was a powerful solubility enhancer. Finally, we replaced the tobacco etch virus (TEV) protease site with a ΔI-CM self-cleavage intein to simplify the purification process. The released soluble TRAIL purity and yield reached 98.4% and 82.1 mg/L in shake flasks, respectively. Thus, the Fh8-ΔI-CM system enhanced target protein solubility by Fh8, enabled on-column tag removal and purification based on Fh8 calcium-binding properties and ΔI-CM self-cleavage properties, and promoted the release of highly active protein with high yield and purity. Overall, our findings suggest that this Fh8-ΔI-CM system could be used as a novel solubility-inducing and purification fusion tag for protein production in E. coli . [ABSTRACT FROM AUTHOR]

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins. • pro-MMP-2 expressed effectively and led to accumulation as inclusion bodies in E. coli. • pro-MMP-2 is easy to be purified to near homogeneity and refolded correctly by rapid dilution. • A cost-effective procedure for obtaining high amounts of functional MMP-2 has been developed. [ABSTRACT FROM AUTHOR]

Melioidosis, an infection caused by the facultative intracellular pathogen Burkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate. B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for the B. pseudomallei infection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle of B. pseudomallei. Further understanding the functional roles of proteins involved in B. pseudomallei TTSS will enable us to dissect the enigma of B. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis of B. pseudomallei, was further characterized using the bioinformatics and molecular approaches. The bipCgene, coding for a putative invasive protein, was first PCR amplified from B. pseudomallei K96243 genomic DNA and cloned into an expression vector for overexpression in Escherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actin in vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by the bipC gene plays a role as an effector involved in the actin binding activity to facilitate internalization of B. pseudomalleiinto the host cells.

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

Alpha-fetoprotein (AFP) is a biomarker that is used to diagnose hepatocellular carcinoma (HCC) and can promote malignancy in HCC. AFP is an important target in the treatment of liver cancer. To obtain enough AFP to screen for AFP inhibitors, we expressed and purified AFP in HEK-293 cells. In the present study, we produced AFP in the cells and harvested highly pure rAFP (or recombinant expression AFP in HEK-293 cells). We also analysed the bioactivity of rAFP and found that rAFP promoted growth of the human HCC cells, antagonize paclitaxel inhibition of HCC cell proliferation, suppress expression of active caspase-3, and promote expression of Ras and survivin. This study provides a method to produce significant amounts of AFP for use in biochemical assays and functional studies and to screen AFP inhibitors for use in HCC therapy. [ABSTRACT FROM AUTHOR]

Ebola virus uses its envelope GP1 and GP2 for viral attachment and entry into host cells. Due to technical difficulty expressing full-length envelope, many structural and functional studies of Ebola envelope protein have been carried out primarily using GP1 lacking its mucin-like domain. As a result, the viral invasion mechanisms involving the mucin-like domain are not fully understood. To elucidate the role of the mucin-like domain of GP1 in Ebola-host attachment and infection and to facilitate vaccine development, we constructed a GP1 expression vector containing the entire attachment region (1–496). Cysteine 53 of GP1, which forms a disulfide bond with GP2, was mutated to serine to avoid potential disulfide bond mispairing. Stable expression clones using codon optimized open reading frame were developed in human 293-H cells with yields reaching ∼25 mg of GP1 protein per liter of spent medium. Purified GP1 was functional and bound to Ebola attachment receptors, DC-SIGN and DC-SIGNR. The over-expression and easy purification characteristic of this system has implications in Ebola research and vaccine development. To further understand the differential expression yields between the codon optimized and native GP1, we analyzed the presence of RNA structural motifs in the first 100 nucleotides of translational initiation AUG site. RNA structural prediction showed the codon optimization removed two potential RNA pseudoknot structures. This methodology is also applicable to the expression of other difficult virus envelope proteins. [ABSTRACT FROM AUTHOR]

Akt, a serine/threonine protein kinase, is often hyper activated in breast and prostate cancers, but with poor prognosis. Allosteric inhibitors regulate aberrant kinase activity by stabilizing the protein in inactive conformation. Several natural compounds have been reported as inhibitors for kinases. In this study, to identify potential natural allosteric inhibitor for Akt1, we generated a seven-point pharmacophore model and screened it through natural compound library. Quercetin-7- O -β- d -glucopyranoside or Q7G was found to be the best among selected molecules based on its hydrogen bond occupancy with key allosteric residues, persistent polar contacts and salt bridges that stabilize Akt1 in inactive conformation and minimum binding free energy during molecular dynamics simulation. Q7G induced dose-dependent inhibition of breast cancer cells (MDA MB-231) and arrested them in G1 and sub-G phase. This was associated with down-regulation of anti-apoptotic protein Bcl-2, up-regulation of cleaved caspase-3 and PARP. Expression of p-Akt (Ser473) was also down-regulated which might be due to Akt1 inhibition in inactive conformation. We further confirmed the Akt1 and Q7G interaction which was observed to have a dissociation constant (K d ) of 0.246 μM. With these computational, biological and thermodynamic studies, we suggest Q7G as a lead molecule and propose for its further optimization. [ABSTRACT FROM AUTHOR]

We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo. For complete details on the use and execution of this protocol, please refer to Crawley et al., 1 Giles et al., 2 and Desbois et al. 3 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). [ABSTRACT FROM AUTHOR]