Protein Expression and Purification

The rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for next-generation antiviral biologics that combine molecular precision with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the receptor-binding domain (RBD) of the spike protein offer a compelling alternative to monoclonal antibodies due to their small size, high thermal stability, and compatibility with microbial expression systems. Here we report the end-to-end development and cGMP production of IPD-52520, a de novo antiviral miniprotein, using an optimized E. coli platform. Two miniprotein candidates, a homotrimeric construct (Trimer is referred to as IPD-52520, 17 kDa) and a tandem fusion (Daisy is referred to as IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media composition, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The Trimer was downselected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetic properties, and higher volumetric manufacturing yields. The optimized process delivers approximately 2 g/L of purified protein at greater than 90% purity. Scale-up from 5 L to 50 L under cGMP conditions demonstrated excellent batch-to-batch reproducibility across six independent batches, supporting nonclinical and Phase 1 clinical supply. Comprehensive biophysical characterization confirmed a well-folded, predominantly alpha-helical trimer (Tm = 73.4 {degrees}C; polydispersity = 1.005) with an intact primary structure and strong target-binding affinity (KD < 1 pM). Real-time stability studies indicate that the drug substance is stable at 2-8 {degrees}C for at least 12 months, with ongoing stability studies. These results demonstrate the feasibility of translating computationally designed antiviral miniproteins into manufacturable biologics and provide a platform applicable to rapid-response therapeutics against current and future pandemic threats.

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a {beta}-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 {micro}M and maximum binding capacity (Bmax) of 144 {micro}mol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 {degrees}C for ONPG and 50 {degrees}C for lactose, and CBE retained >80% activity after 390 min at 45 {degrees}C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 x106 M-1{middle dot}s-1 for ONPG and 4.40 x102 M-1{middle dot}s-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca2+ and Fe2+. These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Background: The N-terminal ectodomain (NTE) of the SARS-CoV-2 membrane (M) glycoprotein is a short, flexible region that remains exposed on the virion surface and exhibits immunogenic potential across multiple coronaviruses. Despite its small size and conformational plasticity, this region contains conserved linear epitopes that may serve as practical surrogates for full-length proteins in serological diagnostics. Objective: To develop and evaluate a synthetic peptide-based diagnostic assay targeting the NTE of the SARS-CoV-2 M protein. Methods: Epitope prediction, peptide synthesis, and antibody affinity assays were performed to design homomultivalent peptide analogs that exploit avidity effects through disulfide polymerization. The resulting peptide antigens were tested in an enzyme-linked immunosorbent assay (ELISA) using clinical samples from RT-PCR-confirmed COVID-19 patients and biobanked controls. Results: The selected peptide analogs (M1, M1i, M1s) corresponded to a conserved surface-exposed motif of the SARS-CoV-2 M protein. Polymeric M1 exhibited a twofold gain in apparent affinity (Kdapp = 4.33 nM) compared with the monomeric form (Kdapp = 8.00 nM). Clinical validation using 1,222 patient samples yielded a sensitivity of 95.26% and specificity of 52.27%, with an overall diagnostic accuracy of 88.70%. Conclusion: The M peptide analogs demonstrate that synthetic peptide antigens can serve as stable, high-sensitivity surrogates for whole-protein assays. This design principle may be applied to other emerging pathogens where rapid assay development and scalability are critical. Keywords: Peptides, Antibodies, COVID-19, Enzyme-Linked Immunosorbent Assay, Protein Binding

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, yet the factors governing reaction longevity remain poorly understood despite their importance for high-throughput applications. Here, the three principal determinants of CFPS performance--DNA template design, reaction composition, and lysate genotype--were systematically optimized to extend reaction lifetime in a 384-well plate format. Different energy regeneration systems were evaluated through real-time pH monitoring and metabolomic analyses to identify the metabolic constraints limiting prolonged protein synthesis. Lysates prepared from engineered Escherichia coli BL21(DE3) strains were further examined to assess the contributions of DNA, RNA, and amino acid stabilization. Systematic optimization of amino acid, nucleoside triphosphate, polyethylene glycol, and lysate concentrations identified DNA template stability and amino acid preservation as the primary factors sustaining CFPS activity. Combining these improvements yielded reactions that remained productive for >14 h and produced 567 {+/-} 64 g mL-1 active deGFP. These findings establish practical strategies for extending CFPS lifetime and improving high-throughput cell-free platforms.

Recombinant proteins have transformative potential in biomedicine, but their production is often costly and carries contamination risks. Plants offer an attractive alternative, with low growth costs and reduced pathogen risk, yet their slow growth cycles limit their use for rapid protein engineering. Here, we establish Marchantia polymorpha, a genetically tractable liverwort with a short life cycle, as a new platform for recombinant protein production. Using stable Agrobacterium-mediated transformation, we expressed an anti-mCherry nanobody fused to the fluorescent protein mTurquoise2 with different purification tags. Expression levels reached up to [~]120 {micro}g/g fresh weight, and nanobody functionality was validated through a microscopy-based bead-binding assay. This yield rivals that of established systems such as Nicotiana benthamiana. Our results position M. polymorpha as a scalable, safe, and efficient chassis for protein engineering, with broad potential for applications in synthetic biology. This work opens the door to exploiting liverwort biotechnology for fast, cost-effective, and biosafe production of valuable recombinant proteins. Graphical Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/733889v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@aabc4aorg.highwire.dtl.DTLVardef@12f9fa4org.highwire.dtl.DTLVardef@199d3e0org.highwire.dtl.DTLVardef@2533a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

For structural-biology and protein-production pipelines, the hardest part of a difficult protein is not the biology -- it is obtaining a well-behaved sample for functional studies. Programs routinely stall at construct design, expression, and purification: deciding where to truncate, which tags to use, how to express, and how to purify so the protein survives concentration and handling. These decisions are still made largely by literature precedent and experimental experience, and they require trial-and-error before arriving at a functional construct for hard targets. We present a prospective, single-pair wet-lab case study testing whether an integrated computational platform can improve these decisions. For human fibroblast growth factor 21 (FGF21) -- a clinically important and stability-challenged metabolic hormone -- we compared two expression constructs produced side by side under the same experimental workflow, using two different design strategies: one designed by a scientist from the literature (reproducing the published core-domain construct, PDB 6M6E), and one designed by the Orbion platform -- an AI, prediction-guided protein-design system (orbion.life) -- which additionally generated the expression and purification protocols (executed scientist-in-the-loop). The platforms construct used an unconventional, longer C-terminal boundary not found in public sequence databases. Since the two constructs differ in more than one feature, we treat them as workflow-level designs throughout. The scientist construct gave a higher initial yield ([~]2.4 xmore protein recovered at affinity capture). The platform-designed construct, however, showed a more favourable downstream developability profile: it concentrated higher (1.4 vs 0.7 mg/mL) while remaining more monodisperse by dynamic light scattering (DLS). The scientist construct, in contrast, aggregated on concentration, so its initial-yield advantage did not survive: in the final concentrated sample the Orbion construct provided the more usable material for downstream studies. Computed for the mammalian host used, the platform had prospectively scored its own design higher (composite 68.7 vs 59.0 for the scientist-designed construct), and its predictions of yield, solubility, and disorder matched the wet-lab outcome. This is a single, deliberately scoped case study, not a population-level benchmark; the two constructs differ in more than one feature, and biological activity was not assayed. Alongside the bottlenecks of this approach discussed here, used as a decision aid, prediction-guided construct and protocol design has the potential to remove costly iteration cycles of protein production campaigns.

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

Cell size is a critical morphological parameter determining cellular functional homeostasis, yet existing large-scale transcriptomic databases lack direct cell size measurement data. By integrating high-resolution immunofluorescence images with transcriptomics, we identified 457 genes significantly correlated with cell area. Based on these findings, we developed an algorithm, Cell Size Score (CSS), to predict cell size from gene expression profiles. Validation across multiple independent datasets, including human cell lines, mouse models, and single-cell spatial transcriptomics, confirmed that CSS accurately predicts cell size. Furthermore, we observed a significant positive correlation between CSS and broad-spectrum chemotherapy drug resistance, suggesting that increased cell volume confers survival advantages to cancer cells. Moreover, CSS analysis of aging revealed sex-dependent, tissue-specific patterns of change, wherein male adipose and cardiac tissues exhibited progressive hypertrophy with age, while female reproductive organs showed significant atrophy. Additionally, CSS significantly increased in skeletal muscle after exercise, indicating that this metric can capture dynamic physiological adaptation processes. This study establishes a bridge between transcriptomics and cell morphology, providing novel insights into retrospectively analyzing the role of cell size in pathological and physiological processes such as cancer and aging using existing omics data, as well as understanding the molecular mechanisms underlying cell size regulation.

Synthetic opioids like fentanyl are a leading cause of overdose mortality. Although the hallmark of fentanyl overdose is ventilatory depression, fentanyl also induces tonic activation of skeletal musculature, including the diaphragm, which may advance progression of overdose towards death. While tonicity may further restrict diaphragmatic contractility, phase-specific dysregulation may also reflect a larger state of discoordination in respiratory control. Using urethane-anesthetized mice exposed to fentanyl, we test the hypothesis that fentanyl-induced diaphragm tonicity results from a loss of coordinated motor activity. Fentanyl produced two distinct phases: an initial phase of maximal ventilatory depression with preserved phasic activity, and a later phase characterized by unstable ventilation that partially rebounds, tonic diaphragmatic activation with loss of inspiratory phase dominance in EMG activity, and diminished bilateral diaphragmatic coordination. Carotid body denervation eliminated tonic activity and expiratory-phase EMG elevation, but it did not prevent hemi-diaphragm discoordination or ventilatory instability. Rhythmic brainstem slice recordings showed that bilateral preBotzinger complex burst-amplitude coupling was disrupted by u-opioid receptor (MOR) agonism. Furthermore, disordered diaphragm activity was reversed by administration of the MOR antagonist, Naloxone. Our findings reframe fentanyl overdose as a temporally evolving syndrome that involves distinct mechanisms to disrupt respiratory motor coordination.

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD) and associated dyslipidemia is a growing health issue that gives rise to cardiovascular risk. Men are more prone to development of MASLD than women. Understanding mechanisms underlying sex differences in MASLD may lead to improved prevention and treatment approaches. Cholesteryl ester transfer protein (CETP) is a lipid transfer protein that shuttles triglycerides and cholesteryl esters between blood lipoproteins and tissues. In this study investigate the impact of hepatic CETP expression on MASLD. Hepatic CETP expression (L-HuCETP) was achieved by injecting liver-targeted CETP-expressing adeno-associated virus into C57BL/6J mice. In females, L-HuCETP improved glucose tolerance, consistent with our prior clamp results in global human CETP transgenic mice. Whereas in males, L-HuCETP worsened glucose metabolism and impaired insulin signaling. Correspondingly, L-HuCETP expression reduced the expression of gluconeogenic pathway genes in females but upregulated these genes in males. In males, L-HuCETP mice exhibited increased hepatic lipid droplet accumulation, lipogenesis proteins and these changes were not observed in females. L-HuCETP expression resulted in sex-specific hepatic responses, with increased expression of inflammation and fibrosis related genes in male, but decreased expression of these genes in females. Mechanistic studies indicate that L-HuCETP had sex specific effects on transcription factors ChREBP and HNF4, which are important for glucose and lipid metabolism. Our studies suggest that sex-specific roles of L-HuCETP with regard to liver metabolic adaptation and MASLD risk in obesity, highlighting CETP-mediated pathways as potential targets for sex-specific precision medicine approaches to improve MASLD.

Skilled control of visually-guided reaching is fundamental for many daily activities. Visual information about hand and target position are used for movement planning and online corrections through rapid visuomotor feedback responses. Such feedback control is generally believed to implicate a single error signal, representing a difference vector between hand and target position. Here, we directly assess whether shared or independent systems serve visually-guided feedback control. We tested whether feedback gains can be independently modulated by hand/cursor and target motion through manipulating the task-relevance of each signal during goal-directed reaching. Our results demonstrate that the gains of visuomotor feedback responses to perturbed hand and target motion can be set independently of one another, at the same time, as a function of task-relevance. By dissociating feedback control of cursor and target signals, our findings support the existence of two independent visuomotor feedback pathways, revealing a more flexible neural architecture for goal-directed action.

Serotonin and dopamine make dissociable contributions to reinforcement learning (RL) sub-components, yet we lack neural biomarkers capable of detecting their differential effects. Here, we report the development of a five-task EEG battery designed to probe these dissociable RL mechanisms. Using a model-free analytical approach in healthy volunteers, we identify distinct neural markers across probabilistic instrumental learning, motivational vigour, Pavlovian-instrumental transfer, reversal learning, and a working memory-RL task. A centro-parietal P300 tracked incremental learning across three paradigms and showed cross-task convergent validity. Readiness potentials and beta suppression indexed value-based motor preparation, while frontal theta captured Pavlovian-instrumental conflict. The largely independent pattern across markers supports the battery's capacity to detect selective pharmacological effects on distinct neural systems.

Memorizing a new phone number or address is hard at first, but becomes easier with repetition, as information shifts from working memory to long-term memory. Here we investigated how repetition affects the storage and transition of different aspects of mnemonic information by comparing univariate neural markers of active object storage with multivariate decoding of memory content. Thirty participants encoded lateralized stimuli from a continuous shape space into memory. Memory items were repeated six times in a row to induce learning. In line with earlier work, EEG recordings revealed that repetition led to a reduction in contralateral delay activity (CDA), a measure of active storage that has been taken to reflect a pointer-like representation of the individual object or its original source. In contrast, shape decoding during the retention and also after an impulse perturbation remained constant across repetitions. These results suggest that learning over repetitions reflects the abolishment of active and individuated object memory representations while passive, source-independent memory representations are retained.

Ethological tasks promise to engage the integrated perception, memory, and decision-making that define natural behavior, yet laboratory implementations are often so sparsified that they may fail to recruit the very cognitive processes of interest. We tested whether increasing environmental complexity in a standard task could expose this hidden cognition. Mice that were already expert hunters in a bare arena were challenged to capture live insect prey in arenas filled with objects that obstruct movement, occlude vision, and offer the prey places to hide. Despite their prior mastery, the added complexity revealed an entire layer of learning that the simple task failed to engage: rather than refining the sensorimotor details of pursuit, mice reorganized how they searched the environment. Across trajectory, kinematic, and object-referenced analyses, learning was expressed predominantly within the search state. To analyze behavior in explicit relation to environmental structure, we developed an open-source framework-a compact ethogram with hierarchical, pose- and object-based classification-that links each action to its environmental context. Unsupervised analyses revealed structured search dynamics across multiple timescales, and a minimal, interpretable agent-based model showed that short-term spatial memory and object-specific value are together sufficient to reproduce the non-random structure of search, including a learned, non-backtracking bias that emerged within the first days of object exposure. Classifiers further showed that mice selectively acquired the object interactions most likely to expose hidden prey. Reproducible with inexpensive materials, the paradigm and its analysis tools offer a sensitive behavioral readout of search, memory, and strategy for studies that conventional low-dimensional assays leave unresolved.

Light is a fundamental regulator of human physiology and behaviour. Whether prior light exposure shapes subsequent higher-order cognition and mood beyond the period of exposure remains unknown. We tested this in a within-subject, randomised crossover experiment in which 24 healthy young adult males completed a multimodal cognitive battery following 2 x15 min of full-spectrum light (FL; median 1,029 melanopic equivalent daylight illuminance [mEDI]) or standard indoor light (SL; median 234 mEDI), with all testing conducted under identical dim illumination. FL improved Digit-Symbol Substitution Test accuracy and promoted digit-directed gaze reallocation, consistent with more efficient associative encoding. On the Balloon Analogue Risk Task, FL reduced reward-seeking behaviour and suppressed backward-referencing gaze transitions linking current and prior-trial reward information. Mood declined following SL but remained stable after FL. Sustained attention, vigilance, and subjective sleepiness were unaffected. Our findings identify pre-task FL exposure as a selective primer of higher-order cognition and mood, independent of alertness.

Studying chess experts helps researchers understand how intensive practice shapes thinking skills. Cognitive flexibility is the ability to adjust thoughts when rules or tasks change. Working memory is the ability to hold and use information over short periods. This study compared cognitive flexibility and working memory precision between adolescent chess players and non-players. Twenty-four professional chess players and twenty-five controls completed two novel behavioral tasks. Chess players showed better accuracy in both tasks than controls. They adapted more efficiently when rules changed during a continuous learning task. They also remembered facial expressions more precisely in a working memory task. Learning rates in the flexibility task did not differ between groups. These results indicate that chess expertise may improve rule-guided flexibility and visual working memory precision in adolescents.

Extremely elongate necks have convergently evolved in several amniote lineages, including both aquatic and terrestrial forms (Fig. 1). The development of such a feature brings with it advantages in obtaining food items, but also biomechanical challenges, such as flexibility, stability, lift, and inertia. In Tanystropheus, a particularly long-necked Triassic archosauromorph, the neck is composed of only 13, mostly extraordinarily elongated and slender cervical vertebrae and accompanying rod-like, overlapping ribs, making it arguably the most extreme example of neck elongation in tetrapod evolution (Fig. 1;1-6). Understanding the function of this remarkable neck provides insights into the limits of neck elongation in amniotes and the evolution of morphological novelties in Triassic reptiles. Here we present the first quantitative biomechanical analysis of the Tanystropheus neck using a digital model based on three-dimensionally preserved bones. We assessed its range of motion (ROM) and performed finite element analysis (FEA) on the individual cervical ribs and the neck model in different configurations. Our results indicate that the neck of Tanystropheus was not extremely stiff, as previously postulated, and the ribs likely did not impair its movements. They transferred tensile forces towards the base of the neck, similar to what hypothesized for sauropods7. This study elucidates the bauplan of an extremely specialized animal and brings us closer to understanding the patterns of achieving neck elongation in vertebrates.

Oxytocin and vasopressin are endogenous bioactive peptides with conserved roles in reproduction and, more recently recognized, in peripheral lipid metabolism. Whether this signaling system also shapes how reproduction declines with age has not been tested in any animal. Here we show that in C. elegans, the oxytocin/vasopressin-like neuropeptide nematocin restrains reproductive output as animals reach mid-life. Nematocin and its two receptors are produced throughout adult life and peak as reproduction begins to wane. Animals lacking receptor signaling produce more offspring in mid-life, an improvement that reflects better egg quality and fertilization rather than improved embryo survival. This benefit is accompanied by changes in intestinal fat metabolism, the worm's equivalent of liver and adipose tissue: nematocin normally limits the activity of a fatty-acid desaturase that is otherwise induced by mating, and it shapes how much yolk reaches developing eggs. The two receptors act through separate routes, one tuning intestinal fat metabolism and the other controlling yolk delivery to the egg. Together, these findings reveal nematocin as a regulator of the intestinal metabolic environment across reproductive age, mirroring the recently described oxytocin-hepatocyte-adipocyte lipid axis in mammals and implicate this conserved signaling system in the coordination of maternal investment during reproductive aging.