分子生物学


分类

现刊
往期刊物
0 Q&A 215 Views Mar 5, 2026

RNA-binding proteins (RBPs) have pleiotropic roles in modulating the physiology of both eukaryotic and prokaryotic cells, enabling them to adapt to environmental variations. The importance of RBPs has led to the development of a variety of methods aiming to identify them. However, most of these approaches have primarily been implemented and optimized in eukaryotic systems. To both uncover novel RBPs involved in Bacillus subtilis sporulation and capture their RNA-binding ability dynamically, we adapted the orthogonal organic phase separation technique (OOPS), which had previously been used in Escherichia coli to reveal its RNA-binding proteome (RBPome). We optimized the UV cross-linking process used to stabilize RNA–protein interactions in vivo and the bacterial lysis process to overcome the robust cell wall of Gram-positive sporulating cells. RNA–protein complexes are then recovered after phase separation steps using guanidinium thiocyanate–phenol–chloroform, and RNA-associated proteins are identified and label-free-quantified by liquid chromatography–mass spectrometry. Collecting samples at various time points during sporulation further enables tracking the dynamics of the RBPome. In addition to being applicable to bacteria and requiring minimal starting material, this method has provided a comprehensive map of the RBPome during sporulation, refining the roles of known factors and revealing new players.

0 Q&A 250 Views Mar 5, 2026

ADGRL4 is an adhesion G protein–coupled receptor (aGPCR) implicated in multiple tumours. In our experience, conventional insect cell-based baculovirus expression systems have not yielded sufficient correctly folded ADGRL4 protein for purification and cryo-electron microscopy (cryo-EM) analysis. Here, we describe aGPCR-HEK, a six-week protocol that establishes stable tetracycline-inducible mammalian HEK293S GnTI- TetR cell lines expressing N-terminally HA- and GFP-tagged aGPCRs. The method comprises lentiviral production in Lenti-X 293T cells, transduction of target adherent HEK293S GnTI- TetR cells, flow cytometry enrichment of uninduced GFP-positive cells displaying leaky expression, adaptation to suspension culture, and large-scale tetracycline induction and harvesting of cells for downstream purification and cryo-EM. The system yields reproducible, milligram-scale quantities of folded aGPCR suitable for structural and biochemical studies.

0 Q&A 570 Views Jan 20, 2026

Although protein–protein interactions (PPIs) are central to nearly all biological processes, identifying and engineering high-affinity intracellular binders remains a significant challenge due to the complexity of the cellular environment and the folding constraints of proteins. Here, we present a two-stage complementary platform that combines magnetic-activated cell sorting (MACS)-based yeast surface display with functional ligand-binding identification by twin-arginine translocation (Tat)-based recognition of associating proteins (FLI-TRAP), a bacterial genetic selection system for efficient screening, validation, and optimization of PPIs. In the first stage, MACS-based yeast display enables the rapid high-throughput identification of candidate binders for a target antigen from a large synthetic-yeast display library through extracellular interaction screening. In the second stage, an antigen-focused library is subcloned into the FLI-TRAP system, which exploits the hitchhiker export process of the Escherichia coli Tat pathway to evaluate binder–antigen binding in the cytoplasm. This stage is achieved by co-expressing a Tat signal peptide–tagged protein of interest with a β-lactamase-tagged antigen target, such that only binder–antigen pairs with sufficient affinity are co-translocated into the periplasm, thus rendering the bacterium β-lactam antibiotic resistant. Because Tat-dependent export requires fully folded and soluble proteins, FLI-TRAP further serves as a stringent in vivo filter for intracellular compatibility, folding, and stability. Therefore, this approach provides a powerful and cost-effective pipeline for discovering and engineering intracellular protein binders with high affinity, specificity, and functional expression in bacterial systems. This workflow holds promise for several applications, including synthetic biology and screening of theragnostic proteins and PPI inhibitors.

0 Q&A 2414 Views Nov 20, 2025

Protein phosphorylation is a dynamic post-translational modification that regulates fundamental processes, including signal transduction, cell proliferation, differentiation, and effector function of immune cells. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is a key mediator of cytokine responses, essential for maintaining immune cell homeostasis and determining cell fate across diverse immune subsets. Dysregulation of JAK/STAT signaling has been linked to a broad spectrum of pathologies, including monogenic immune disorders, autoimmunity, and cancer. Platforms facilitating single-cell analysis of protein phosphorylation offer the ability to reveal subtle signaling defects and dissect the pleiotropy in cellular composition and phosphorylation status, providing insights into immune phenotype and function, while identifying potential therapeutic targets. While an application of cytometry-by-time-of-flight, termed phospho-CyTOF, has proven invaluable for studying protein phosphorylation in cryopreserved peripheral blood mononuclear cells (cPBMCs), its application is limited by cell loss and signaling artifacts stemming from isolation and cryopreservation. Conversely, whole blood (WB) approaches, preserving the native immune cell composition and signaling context, offer a more physiological representation but necessitate robust and consistent protocols for broad application. Herein, we present optimized dual phospho-CyTOF workflows tailored for both cPBMCs and whole blood, building upon established protocols for cytokine stimulation of both samples. These workflows facilitate comprehensive, high-dimensional profiling of JAK/STAT signaling in response to pleiotropic cytokines such as Type I interferons (IFN-α), Type II interferons (IFN-γ), and Interleukin-21 (IL-21). By leveraging CyTOF's capacity for high-dimensional profiling using pure heavy metal–labeled antibodies, these protocols aim to identify pathway-specific alterations in STAT phosphorylation across major immune subsets that may be overlooked by traditional flow cytometry. Together, these optimized dual workflows provide scalable, translationally relevant tools for dissecting the subtle and differential JAK/STAT-driven immune responses in both clinical and research settings, while also being compatible with the simultaneous assessment of crosstalk with alternative immune cell signaling pathways.

0 Q&A 2522 Views Aug 20, 2025

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, producing ATP in the final step of glycolysis. Unlike other isoforms, PKM2 is uniquely regulated, shifting between active tetramers and less active dimers to balance energy production with biosynthetic demands. This flexibility is exploited in cancer cells to support the Warburg effect and anabolic growth. Additionally, PKM2 can translocate to the nucleus and act as a transcriptional co-activator, influencing gene expression and tumor progression. To facilitate functional studies of PKM2, we present a robust and reproducible protocol for its expression, purification, and enzymatic characterization. PKM2 is expressed in E. coli and purified via Ni-NTA affinity and size-exclusion chromatography to ensure high purity and proper folding. Enzymatic activity is measured using a lactate dehydrogenase (LDH)-coupled assay that tracks NADH oxidation at 340 nm, allowing sensitive kinetic analysis under various conditions, including different PEP concentrations, pH levels, and presence of the allosteric activator fructose-1,6-bisphosphate (FBP). This non-radioactive, high-resolution method is suitable for analyzing PKM2 regulation, post-translational modifications, and mutant variants, as well as for screening potential therapeutic modulators, providing a valuable tool for cancer metabolism research.

0 Q&A 2607 Views Jul 20, 2025

Accurate measurement of protein translation rates is crucial for understanding cellular processes and disease mechanisms. However, existing methods for quantifying translation rates in yeast cells are limited. Here, we present a streamlined protocol for measuring protein translation rates in Saccharomyces cerevisiae using the methionine analog L-azidohomoalanine (AHA), which is the L isoform of this synthetic amino acid, and fluorophore-labeled alkyne dye-based Click chemistry. Our method involves incorporating AHA into newly synthesized proteins, followed by detection using confocal microscopy, flow cytometry, and SDS-PAGE. We validated our protocol by measuring translation rates under various stress conditions, including heat stress, endoplasmic reticulum (ER) stress induced by tunicamycin, and translation inhibition by cycloheximide. Confocal microscopy revealed differential AHA incorporation and fluorescence intensity across conditions. Flow cytometry quantitatively confirmed significant increases in translation rates under heat stress and decreases under ER stress compared to unstressed conditions at 6 and 24 h post-treatment. Imaging of gels under fluorescence detectors following SDS-PAGE further visualized newly synthesized proteins, with no detectable translation after cycloheximide treatment. Our protocol offers enhanced precision and selectivity compared to existing methods for mammalian cells and represents the first standardized approach for measuring translation rates in yeast. Despite limitations in required specialized equipment and expertise, this method holds promise for diverse applications in biotechnology and biomedical research, enabling investigations into protein synthesis regulation in yeast systems.

0 Q&A 1785 Views Jul 20, 2025

Science self-efficacy describes the confidence individuals have in their ability to accomplish specific scientific practices. Self-efficacy is one factor linked to success and persistence within STEM fields. The purpose of this protocol is to provide research laboratories with effective methods for teaching and mentoring new students in molecular biology, specifically in the synthesis of virus-like particles (VLPs) derived from bacteriophages. VLPs are multivalent nanoparticle structures that can be utilized in multiple biomedical applications, including platforms for vaccine and drug delivery. Production of bacteriophage VLPs using bacterial expression systems is feasible in most laboratory settings. However, synthesizing and characterizing VLPs can be challenging for new researchers, especially those with minimal laboratory experience or a lack of foundational knowledge in molecular biology. To address this, a multi-phase training protocol was implemented to train new students in VLP synthesis, purification, and characterization. This model was optimized for training numerous high school and undergraduate students. By implementing this multi-phase methodology, the students’ confidence in their abilities to perform specific tasks increased and likely enhanced their persistence in STEM.

0 Q&A 2795 Views Jul 20, 2025

This manuscript details protocols for the ZnCl2 precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl 2 at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, such as sodium dodecyl sulfate (SDS), Triton X-100, and urea, at high concentrations in solution from protein solutions prior to trypsin digestion. It is a practical, robust, and cost-effective approach for proteomic sample preparation. It has been observed that 90.2% of the proteins can be recovered from lysates by incubating with an equal volume of ZPB at room temperature for 10 min. In 1 h of data-dependent acquisition (DDA) analysis on an Exploris 480, 4,037 proteins and 25,626 peptides were quantified from 1 μg of mouse small intestine proteins, reaching a peak of 4,500 proteins and up to 30,000 peptides with 5 μg of input. Additionally, ZASP outperformed other common sample preparation methods such as sodium deoxycholate (SDC)-based in-solution digestion, acetone precipitation, filter-aided sample preparation (FASP), and single-pot, solid-phase-enhanced sample preparation (SP3). It demonstrated superior performance in protein (4,456 proteins) and peptide identification (29,871 peptides), lower missing cleavage rates (16.3%), and high reproducibility (Pearson correlation coefficients of 0.96 between replicates) with similar protein distributions and cellular localization patterns. Significantly, the cost of ZASP per sample with 100 μg of protein as input is lower than 30 RMB, including the expense of trypsin.

0 Q&A 2338 Views Jul 20, 2025

Cathepsin L (CTSL), a lysosomal cysteine protease belonging to the papain-like protease family, is primarily involved in intracellular protein degradation, antigen processing, and extracellular matrix remodeling. It plays critical roles in pathological conditions, including cancer metastasis, neurodegenerative disorders, and viral infection, due to dysregulated activity or overexpression. Thus, inhibitors targeting CTSL are under investigation for therapeutic applications. Current approaches for identifying CTSL inhibitors predominantly rely on fluorescence-labeled substrates, fluorescence resonance energy transfer (FRET), and cell-based screening assays. Here, we applied the principle of fluorescence polarization (FP) to the detection of substrate cleavage activity by CTSL through changes in millipolarization unit (mp) values and established a cost-effective, quantitative, reagent- and time-saving inhibitor high-throughput screening (HTS) assay. We also provide detailed steps for the expression and purification of highly active CTSL from eukaryotic cells, which lays a solid foundation for the FP-based assay. A key advantage of this assay lies in its reduced susceptibility to fluorescence interference, as the fluorescein isothiocyanate (FITC) fluorophore exhibits high quantum efficiency with an emission peak at 535 nm—a wavelength range distinct from most naturally occurring fluorescent molecules. The assay’s adaptability to reaction time, temperature, and dimethyl sulfoxide (DMSO) concentration minimizes false-positive or false-negative results caused by minor experimental inconsistencies, streamlining the screening process. Furthermore, the protocol requires fewer operational steps, reduced incubation time, and lower quantities of CTSL and substrates compared to conventional methods. This rapid, cost-effective, and scalable approach aligns well with the demands of HTS platforms.

0 Q&A 2266 Views Jul 5, 2025

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-32P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.