Protein Expression and Purification

Recombinant protein purification from E. coli frequently requires screening various affinity tags and variations to optimize yield and purity. However, classical cloning methods are limited in throughput and modularity, which is circumvented by GoldenGate cloning. While GreenGate cloning, a GoldenGate variant, is widely used in plant research, it lacks compatibility with E. coli expression vectors. Here, we introduce a comprehensive, GreenGate-compatible vector toolkit for efficient and versatile assembly of three modules, for N- and C-terminal tagging of a protein of interest into an IPTG-inducible E. coli expression vector. To allow versatility, this toolkit contains diverse affinity tags, with or without HRV3C protease cleavage sites. Moreover, we included plasmids for the homemade low-cost production of this protease. This GreenGate-compatible toolkit allows efficient combinations of different tags and eliminates the need for re-cloning modules between plant and bacterial systems, streamlining the workflow for recombinant protein production, especially in plant research.

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complimentary determining region 2 (CDR2) of a soluble, clinically validated nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered CALR-Nb02 by adapting the core of Cal3 to a partial consensus framework sequence of stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a predominantly monomeric, soluble protein that exhibits an increased thermal stability and a higher calreticulin binding affinity (KD: 25-50 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody, and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.

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.

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.

Phycobiliproteins form oligomeric assemblies essential for photosynthetic light harvesting. Here, we engineered phycocyanin (TeCPC) and allophycocyanin (TeAPC) from Thermosynechococcus elongatus to stabilize defined trimers by inhibiting hexamer formation. Structure-guided substitutions at conserved glycine residues (TeCPC G29R, TeAPC G21R) introduce steric hindrance at the hexamer interface. Recombinant expression in Escherichia coli produced holoproteins with native-like chromophorylation. Biophysical and structural analyses confirmed homogeneous trimer formation and absence of higher-order assemblies. Thermal measurements indicated cooperative unfolding, supporting structural uniformity. These engineered trimers provide robust models for studying energy transfer in phycobiliproteins.

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.

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.

We previously generated a mouse monoclonal antibody, N179, against the SARS-CoV-2 nucleocapsid (N) protein and developed a colloidal gold-based immunochromatographic test strip. This assay achieved a detection limit of 2 ng/mL and displayed 98% concordance with RT-qPCR results. However, the precise epitope recognized by mAb N179 had not been defined. Using a panel of GST-fused N protein truncation fragments, we mapped the linear B-cell epitope recognized by mAb N179 to the flexible C-terminal tail of the N protein by Western blotting and ELISA. The minimal binding motif required for mAb N179 recognition was identified as 390QTVTLL395. Multiple sequence alignment of 11 representative SARS-CoV-2 lineages, including Alpha, Beta, Gamma, Delta, and Omicron subvariants BA.1, BA.2, and BA.3.2, revealed that this epitope was completely conserved across all variants analyzed. Stringent local pairwise alignment analysis using EMBOSS WATER further showed that the 390QTVTLL395 motif achieved a perfect 6/6 match exclusively in SARS-CoV-2; no identical sequence was detected in the seven common human coronaviruses, four influenza viruses, or five bat coronaviruses examined. Structural prediction analyses indicated that this region is surface-exposed and possesses a strong linear B-cell epitope propensity. Together, these findings identify 390QTVTLL395 as a specific molecular signature of SARS-CoV-2 among the viruses analyzed. Our results provide an epitope-level explanation for the sustained diagnostic reliability of the mAb N179-based assay against emerging variants, clarify the molecular basis for its lack of cross-reactivity, and may inform the rational design of SARS-CoV-2 diagnostics targeting conserved, mutation-resistant epitopes. ImportanceWe identified the exact nucleocapsid protein epitope recognized by monoclonal antibody N179, a diagnostic antibody used in a colloidal gold rapid assay. The identified 390QTVTLL395 motif at residues 390 to 395 was conserved among the SARS-CoV-2 variants analyzed and was not present as an identical continuous sequence in the related respiratory viruses examined. This work supports precise epitope mapping as a useful strategy for evaluating and revalidating diagnostic antibodies as respiratory viruses evolve.

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.

1.Virus-like particles (VLPs) formed from the murine polyomavirus major capsid protein VP1 are widely used as vaccine antigens and are being explored as nucleic acid and drug delivery vehicles. However, the factors controlling distinct VP1 capsid morphologies remain unclear. We investigated in vitro VP1 assembly with tRNA across NaCl concentrations of 0.15-1.0 M and tRNA mass ratios of 1:1-1:80 (w/w) using SEC-HPLC, transmission electron microscopy, and dynamic light scattering. Two competing assembly pathways were identified. At low ionic strength ([&le;]0.15 M NaCl), nucleic acid-templated assembly produced compact, tRNA-filled T=1 VLPs ([~]28-30 nm). Assembly was maximal at tRNA ratios of 1:10-1:20, whereas excess or insufficient tRNA reduced yields. Increasing NaCl to 0.30 M lowered T=1 yields by 68-97%, and no T=1 particles were detected at [&ge;]0.5 M NaCl. Conversely, high ionic strength ([&ge;]0.5 M NaCl) promoted template-independent formation of hollow T=7 VLPs ([~]55-60 nm). T=7 assembly was inhibited by tRNA and was highest without nucleic acid. At 1.0 M NaCl, reducing the tRNA ratio from 1:40 to 1:80 increased T=7 yield more than 11-fold, while removing tRNA produced the greatest assembly efficiency. Kinetic analyses further showed that VP1 concentration and ionic strength regulate nucleation and assembly rate in the template-driven pathway. These findings show that electrostatic interactions govern pathway selection between tRNA-templated T=1 assembly and salt-driven T=7 self-assembly, providing practical guidance for controlling capsid morphology and cargo loading in VLP-based applications.

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.

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

BackgroundRecombinant expression of the SARS-CoV-2 receptor-binding domain (RBD) is essential for vaccine development, serological diagnostics, and mechanistic studies. Primary human fibroblasts offer physiologically relevant protein folding and post-translational modification, yet their short lifespan limits scalable production. We used an immortalized human splenic fibroblast cell line to stably express RBD-sfGFP for longitudinal characterization and downstream studies. MethodsImmortalized human primary splenic fibroblasts were transfected by electroporation with a plasmid encoding SARS-CoV-2 RBD fused to superfolder GFP (sfGFP), with a neomycin resistance cassette (neoR) for G418 selection. Four independent G418-resistant cultures (n=4), designated HPSF-IM-RBD-BHSKPU T1-T4, were established from distinct selection flasks. Based on previous screenings, two cultures (T1, T3) were monitored for 98 days (14 passages, P1-P14); two cultures (T2, T4) were monitored for 42 days (6 passages, P1-P6). RBD-sfGFP expression was assessed by fluorescence microscopy at 7-day intervals. For each timepoint, 2 fields were imaged and analyzed for relative fluorescence intensity (normalized to global maximum = 100%) and mean fluorescence intensity (MFI, normalized to global maximum = 100%). Coefficient of variation (CV), linear regression, and Pearson correlation were calculated. ResultsAll four cultures exhibited robust GFP fluorescence, confirming stable transgene retention. Expression ranking: T1 (93.1% +/- 3.6%) > T3 (89.2% +/- 3.4%) > T2 (84.2% +/- 3.2%) > T4 (79.7% +/- 3.9%). Long-term cultures T1 and T3 retained [~]100% of Day 7 signal at Day 98 (T1: 100.7%; T3: 100.0%). Expression exhibited passage-dependent oscillation rather than progressive silencing. CV increased over time in T1 (1.5% -> 8.5%), indicating growing inter-cellular heterogeneity. A strong positive correlation between fluorescence and MFI (Pearson r = 0.823, p = 7.44 x 10-11) suggested coherent population-level regulation. ConclusionsHPSF-IM-RBD-BHSKPU cells stably retain RBD-sfGFP expression for over 3 months, validating their utility as a recombinant protein production platform. However, oscillatory dynamics and increasing heterogeneity are consistent with position-effect variegation at distinct integration loci. Consequently, early passages (P1-P4) are optimal for applications requiring maximal uniformity. Ultimately, these cells provide a practical tool for RBD production and a valuable model for studying epigenetic regulation of transgene expression in human primary fibroblast backgrounds.

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

AbstractMetarhizium anisopliae is an important entomopathogenic fungi used in biological control of agricultural pests, but its commercial application is limited by relatively slow host mortality. This study aimed to engineer a chimeric protease (cPr1A) with enhanced binding affinity and protease activity against insect cuticle. We hypothesized that stronger cuticle binding would increase local enzyme concentration at the cuticle surface and thereby enhance cuticle degradation. To achieve this, the Bombyx mori chitin-binding domain (BmCBD) was fused to the C-terminus of the Pr1A protease from M. anisopliae. Recombinant Pr1A and cPr1A were expressed in Escherichia coli, purified by Ni-NTA affinity chromatography. Binding and protease activity were assayed in triplicate using Samia ricini cuticle powder as substrate. Results are presented as mean +/- SEM. The chimeric protease cPr1A showed a 28.9% increase in cuticle binding compared to wild-type Pr1A (15.81 +/- 1.97 vs. 12.27 +/- 2.13 g bound protein/mg cuticle powder; p < 0.002) and a 35% increase in protease activity (0.343 +/- 0.08 U/mg vs. 0.254 +/- 0.06 U/mg; p < 0.03). These results indicate that cPr1A is a promising candidate for overexpression in M. anisopliae to enhance cuticle degradation and potentially improve fungal virulence against insect pests.

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process -1,4- and -1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as -1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel -1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves -1,2-linkages via an anomer-inverting mechanism, with a distinct preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 [A]) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts a (/{beta})6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses -1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare -glucans.

Introduction: Synthetic oligopeptides provide a rapid and cost-efficient approach to developing antibodies and diagnostics for emerging viral variants. Methods: This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC). Binding affinity was computationally estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), while experimental validation was performed using enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical diagnostic accuracy testing was done using plasma samples from RT-PCR-confirmed COVID-19 patients and pre-COVID-19 controls. Results: S621 demonstrated nanomolar binding affinity (Kdapp = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). Diagnostic evaluation yielded a sensitivity of 89.92% and specificity of 27.79%, corresponding to an overall accuracy of 71.79%. Discussion: These findings demonstrate that a single synthetic peptide derived from a conserved spike subdomain can function as a high-affinity surrogate for full-length antigens, supporting its potential application in rapid peptide-based immunodiagnostics.

Bacterial microcompartments (MCPs) are versatile proteinaceous organelles that compartmentalize metabolic pathways, offering promising scaffolds for synthetic biology and metabolic engineering. However, designing customized nanobioreactors requires distinguishing structurally indispensable shell proteins from those that can be modified or deleted to tune shell permeability without disrupting core organelle assembly. In this study, we performed a systematic biophysical and metabolic characterization of the hexameric shell protein PduU to evaluate its potential as a modular platform for synthetic organelle engineering. We tested whether deleting pduU or selectively truncating its N-terminal {beta}-barrel domain preserves shell assembly, metabolite flux, and intermediate confinement. Our results demonstrate that PduU modifications alter shell permeability while fully maintaining organelle structural integrity, monodispersity, and electrostatic colloidal stability. Crucially, this modulation in permeability redirects internal metabolic flux toward the energy-generating propionate pathway, resulting in elevated cell biomass and significantly increased yields of propionate, an economically vital industrial platform chemical. By establishing that PduU is a non-essential structural component whose modification tunes small-molecule flux, this work highlights PduU as a flexible locus for shell engineering, providing a scalable strategy for biomanufacturing of high-value bio-based products in tailor-made MCP nanobioreactors.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

The successful translation of rationally designed HIV-1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical-grade quality standards. We developed and scaled a robust, cGMP-compliant process for N332-GT5 gp140, a germline-targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first-in-human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap-In(R) transposon technology, we established a production clone exhibiting high-titer expression (>200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled efficiently from Ambr(R) 250 miniature bioreactors to 200-L single-use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three-step purification strategy--affinity capture, multimodal polishing, and viral clearance- yielded >99% trimeric purity with preserved quaternary structure and native-like antigenicity. Orthogonal LC-MS analyses confirmed site-specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18-log and 11-log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV-1 envelope immunogens, advancing the field toward rational vaccine design based on germline-targeting principles.