SCF-ChemBio: Chemical Biology Tour de France.

Chemical biology is a steadily growing field that has traditionally struggled to clearly define its boundaries in a short sentence. However, it can be stated that through the development of chemical and physicochemical tools, concepts and methods, chemical biology aims to address or stimulate biological questions at the molecular level in living organisms. Chemical biologists design and develop molecular tools that can probe or modulate biological processes, in order to understand their function, and sometimes to modify it for specific applications, but also to observe and analyze these tools in complex biological environments. Essentially positioned as a fundamental approach, chemical biology often remains very close to potential applications as it builds molecular objects capable of reacting to a significant biological stimulus. Chemical biology therefore finds natural development in fields such as health for the design of drugs and diagnostic systems or the environment for applications in crop science and ecology.

REPRISAL: mapping lignification dynamics using chemistry, data segmentation, and ratiometric analysis.

This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called "REPRISAL" for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G*, and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labeling strategy based on the sequential use of three main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence segmentation algorithm is developed that assigns fluorescent image pixels to three distinct cell wall zones corresponding to cell corners, compound middle lamella and secondary cell walls. The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method [RM] 1) and proportions (RM 2) within the different cell wall zones. We first describe the use of this methodology to map developmentally related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × Populus alba), flax (Linum usitatissimum), and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into noncellulosic cell wall polymers.

A novel C-domain-dependent inhibition of the rainbow trout CMP-sialic acid synthetase activity by CMP-deaminoneuraminic acid.

The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by 31P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

To View Your Biomolecule, Click inside the Cell.

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

The in Vitro Antiplasmodial and Antiproliferative Activity of New Ferrocene-Based α-Aminocresols Targeting Hemozoin Inhibition and DNA Interaction.

The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α-aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α-aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and -resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple-negative breast cancer cell line. Indication of cross-resistance was absent for the compounds evaluated against the multi-resistant Dd2 strain. Structure-activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α-carbon and NH group of the α-amino-o-cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor-groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer.

Exploring the Potential of ß-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator ß-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of ß-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of ß-catenin in HeLa cells. The changes in O-GlcNAcylation of ß-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.

OGT Controls the Expression and the Glycosylation of E-cadherin, and Affects Glycosphingolipid Structures in Human Colon Cell Lines.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation, and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affects overall N- and O-glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

MicroPlate Sialyltransferase Assay: A Rapid and Sensitive Assay Based on an Unnatural Sialic Acid Donor and Bioorthogonal Chemistry.

Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate assay based on the use of the unnatural CMP-SiaNAl donor substrate. In this assay, an appropriate acceptor glycoprotein is coated on the bottom of 96-well plate and the sialyltransferase activity is assessed using CMP-SiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an azido-biotin probe via CuAAC and an antibiotin-HRP conjugated antibody is then used to quantify the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase inhibitory effect.

Chemical glycomics enrichment: imaging the recycling of sialic acid in living cells.

The development of metabolic oligosaccharide engineering (MOE) over the past two decades enabled the bioimaging studies of glycosylation processes in physio-pathological contexts. Herein, we successfully applied the chemical reporter strategy to image the fate of sialylated glycoconjugates in healthy and sialin-deficient patient fibroblasts. This chemical glycomics enrichment is a powerful tool for tracking sialylated glycoconjugates and probing lysosomal recycling capacities. Thus, such strategies appear fundamental for the characterization of lysosomal storage diseases.

The Many Ways by Which O-GlcNAcylation May Orchestrate the Diversity of Complex Glycosylations.

Unlike complex glycosylations, O-GlcNAcylation consists of the addition of a single N-acetylglucosamine unit to serine and threonine residues of target proteins, and is confined within the nucleocytoplasmic and mitochondrial compartments. Nevertheless, a number of clues tend to show that O-GlcNAcylation is a pivotal regulatory element of its complex counterparts. In this perspective, we gather the evidence reported to date regarding this connection. We propose different levels of regulation that encompass the competition for the nucleotide sugar UDP-GlcNAc, and that control the wide class of glycosylation enzymes via their expression, catalytic activity, and trafficking. We sought to better envision that nutrient fluxes control the elaboration of glycans, not only at the level of their structure composition, but also through sweet regulating actors.

One, Two, Three: A Bioorthogonal Triple Labelling Strategy for Studying the Dynamics of Plant Cell Wall Formation In Vivo.

Reported herein is an in vivo triple labelling strategy to monitor the formation of plant cell walls. Based on a combination of copper-catalysed alkyne-azide cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), and Diels-Alder reaction with inverse electronic demand (DARinv ), this methodology can be applied to various plant species of interest in research. It allowed detection of the differential incorporation of alkynyl-, azido-, and methylcyclopropenyl-tagged reporters of the three main monolignols into de novo biosynthesized lignin in different tissues, cell types, or cell wall layers. In addition, this triple labelling was implemented with different classes of chemical reporters, using two monolignol reporters in conjunction with alkynylfucose to simultaneously monitor the biosynthesis of lignin and non-cellulosic polysaccharides. This allowed observation of their deposition occurring contemporaneously in the same cell wall.

A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus.

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

Probing the CMP-Sialic Acid Donor Specificity of Two Human ß-d-Galactoside Sialyltransferases (ST3Gal†I and ST6Gal†I) Selectively Acting on O- and N-Glycosylproteins.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human ß-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids.

Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved 31P phosphorus nuclear magnetic resonance (31P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. 31P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.

The antimalarial ferroquine is an inhibitor of hepatitis C virus.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. CONCLUSION: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.