Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.

Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Modified streptavidin-biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤ 35, and Ct ≤ 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

Active targeting tumor therapy using host-guest drug delivery system based on biotin functionalized azocalix[4]arene.

Host-guest drug delivery systems (HGDDSs) provided a facile method for incorporating biomedical functions, including efficient drug-loading, passive targeting, and controlled drug release. However, developing HGDDSs with active targeting is hindered by the difficult functionalization of popular macrocycles. Herein, we report an active targeting HGDDS based on biotin-modified sulfonated azocalix[4]arene (Biotin-SAC4A) to efficiently deliver drug into cancer cells for improving anti-tumor effect. Biotin-SAC4A was synthesized by amide condensation and azo coupling. Biotin-SAC4A demonstrated hypoxia responsive targeting and active targeting through azo and biotin groups, respectively. DOX@Biotin-SAC4A, which was prepared by loading doxorubicin (DOX) in Biotin-SAC4A, was evaluated for tumor targeting and therapy in vitro and in vivo. DOX@Biotin-SAC4A formulation effectively killed cancer cells in vitro and more efficiently delivered DOX to the lesion than the similar formulation without active targeting. Therefore, DOX@Biotin-SAC4A significantly improved the in vivo anti-tumor effect of free DOX. The facilely prepared Biotin-SAC4A offers strong DOX complexation, active targeting, and hypoxia-triggered release, providing a favorable host for effective breast cancer chemotherapy in HGDDSs. Moreover, Biotin-SAC4A also has potential to deliver agents for other therapeutic modalities and diseases.

CYP1B1 affects the integrity of the blood-brain barrier and oxidative stress in the striatum: An investigation of manganese-induced neurotoxicity.

AIMS: Excessive influx of manganese (Mn) into the brain across the blood-brain barrier induces neurodegeneration. CYP1B1 is involved in the metabolism of arachidonic acid (AA) that affects vascular homeostasis. We aimed to investigate the effect of brain CYP1B1 on Mn-induced neurotoxicity. METHOD: Brain Mn concentrations and α-synuclein accumulation were measured in wild-type and CYP1B1 knockout mice treated with MnCl2 (30 mg/kg) and biotin (0.2 g/kg) for 21 continuous days. Tight junctions and oxidative stress were analyzed in hCMEC/D3 and SH-SY5Y cells after the treatment with MnCl2 (200 µM) and CYP1B1-derived AA metabolites (HETEs and EETs). RESULTS: Mn exposure inhibited brain CYP1B1, and CYP1B1 deficiency increased brain Mn concentrations and accelerated α-synuclein deposition in the striatum. CYP1B1 deficiency disrupted the integrity of the blood-brain barrier (BBB) and increased the ratio of 3, 4-dihydroxyphenylacetic acid (DOPAC) to dopamine in the striatum. HETEs attenuated Mn-induced inhibition of tight junctions by activating PPARγ in endothelial cells. Additionally, EETs attenuated Mn-induced up-regulation of the KLF/MAO-B axis and down-regulation of NRF2 in neuronal cells. Biotin up-regulated brain CYP1B1 and reduced Mn-induced neurotoxicity in mice. CONCLUSIONS: Brain CYP1B1 plays a critical role in both cerebrovascular and dopamine homeostasis, which might serve as a novel therapeutic target for the prevention of Mn-induced neurotoxicity.

Biotin-new indocyanine green conjugate: Synthesis, in vitro photocytotoxicity and in vivo biodistribution.

New indocyanine green (ICG) (IR820) is one of the ICG derivatives and attracts increasing attention for cancer management. However, the unsatisfactory tumor targeting ability of IR820 significantly limits its applications for cancer theranostics. Biotin receptor is overexpressed on the membrane of various tumor cells and biotin modified nanocarriers have been reported to enhance the tumor targeting ability on several tumor types. In this work, biotin-new ICG conjugate (Biotin-SS-IR820) was prepared for tumor-targeted IR820 delivery. Biotin and IR820 were coupled through cystamine. The synthesized Biotin-SS-IR820 was characterized by 1 H NMR, FT-IR and HRMS. The in vitro singlet oxygen generation study shows that Biotin-SS-IR820 exhibits similar singlet oxygen generation as compared to IR820 upon 660 nm laser irradiation (0.8 W/cm2 ). The cellular uptake study shows that Biotin-SS-IR820 shows enhanced cellular uptake amount as compared to IR820 on 4T1 cells. As a result, Biotin-SS-IR820 displays enhanced in vitro photodynamic therapeutic effect against 4T1 cells as compared to IR820. In in vivo biodistribution study, Biotin-SS-IR820 shows enhanced tumor accumulation as compared to IR820. Biotin-SS-IR820 developed in this work shows promising prospects for targeted delivery of IR820 to biotin receptor overexpressed tumors.

Heterologous antigen selection of chicken single-chain variable fragments against thiamethoxam.

Single-chain variable fragments (scFvs) are valuable in the development of immunoassays for pesticide detection. In this study, scFvs specific to thiamethoxam (Thi) were successfully isolated from a library generated by chicken immunization through heterologous coating selection. These scFvs were subsequently expressed with fusion with an Avi tag and alkaline phosphatase. After combination and optimization, a scFv-biotin based enzyme linked immunosorbent assay (ELISA) was developed for the detection of Thi, demonstrating an impressive half-maximum signal inhibition concentration (IC50) of 30 ng mL-1 and a limit of detection (LOD) of 1.8 ng mL-1. The immunoassay exhibited minimal cross-reactivity with other neonicotinoid insecticides, except for 7.5% for imidacloprid and 6.7% for imidaclothiz. The accuracy of the assay was confirmed by testing spiked samples of apple, pear, cabbage, and cucumber, which resulted in average recoveries ranging between 82% and 119%, closely aligning with the results obtained through high-performance liquid chromatography. Therefore, the chicken scFv-biotin based assay showed promise as a high-throughput screening tool for Thi in agricultural samples.

Enhanced fatty acid storage combined with the multi-factor optimization of fermentation for high-level production of docosahexaenoic acid in Schizochytrium sp.

Schizochytrium sp. hasreceived much attention for itsability to synthesize and accumulate high-level docosahexaenoic acid (DHA), which can reach nearly 40 % of total fatty acids. In this study, the titer of DHA in Schizochytrium sp. was successfully improved by enhancing DHA storage through overexpressing the diacylglycerol acyltransferase (ScDGAT2C) gene, as well as optimizing the supply of precursors and cofactors required for DHA synthesis by response surface methodology. Notably, malic acid, citric acid, and biotin showed synergistic and time-dependent effects on DHA accumulation. The maximum lipid and DHA titers of the engineered Schizochytrium sp. strain reached 84.28 ± 1.02 g/L and 42.23 ± 0.69 g/L, respectively, with the optimal concentration combination (1.62 g/L malic acid + 0.37 g/L citric acid + 8.28 mg/L biotin) were added 48 h after inoculation. This study provides an effective strategy for improving lipid and DHA production in Schizochytrium sp.

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase.

G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3' splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation.

Polydopamine (pDA) is a valuable material with wide-ranging potential applications. However, the complex and debated nature of dopamine polymerization complicates our understanding. Specifically, the impact of foreign substances, especially proteins, on pDA formation adds an additional layer of subtlety and complexity. This study delves into specific surface features of proteins that predominantly shape their impact on dopamine polymerization. Notably, the biotin-binding site emerges as a critical region responsible for the pronounced inhibitory effect of avidin and neutravidin on the dopamine polymerization process. The binding of biotin successfully mitigates these inhibitory effects. Moreover, several nucleases demonstrated a significant hindrance to pDA formation, with their impact substantially alleviated through the introduction of DNA. It is speculated that hydrogen bonding, electrostatic, cation-π, and/or hydrophobic interactions may underlie the binding between protein surfaces and diverse oligomeric intermediates formed by the oxidation products of dopamine. Additionally, we observed a noteworthy blocking effect on the dopamine polymerization reaction induced by erythropoietin (EPO), a glycoprotein hormone known for its role in stimulating red blood cell production and demonstrating neuroprotective effects. The inhibitory influence of EPO persisted even after deglycosylation. These findings not only advance our comprehension of the mechanisms underlying dopamine polymerization but also provide strategic insights for manipulating the reaction to tailor desired biomaterials.

Climbing into their Skin to Understand Contextual Protein-Protein Associations and Localizations: Functional Investigations in Transgenic Live Model Organisms.

Borrowing some quotes from Harper Lee's novel "To Kill A Mockingbird" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.

Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.

ß-barrel membrane proteins play a crucial role in bacterial pathogenesis and antibiotic resistance, making them a prime focus for the development of new antibiotics and therapeutics. However, their inherent hydrophobic nature and limited presence pose challenges for their high-throughput characterization using conventional methods. In this context, we present a simple but efficacious approach using peptidisc, a membrane mimetic, to overcome the low abundance and hydrophobicity of these proteins. Our methodology, illustrated here using Escherichia coli (E. coli) as a model organism, covers the entire process from outer membrane fraction preparation to data analysis. This detailed protocol outlines the purification of a diverse collection of ß-barrel membrane proteins, rendering them water-soluble and readily amenable to mass spectrometry and downstream drug screening strategies.