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Background: The immunosuppressive microenvironment negatively affects the efficacy of chimeric antigen receptor T (CAR-T) cells in solid tumors. Fusion protein that combining extracellular domain of inhibitory checkpoint protein and the cytoplasmic domain of stimulatory molecule may improve the efficacy of CAR-T cells by reversing the suppressive signals., Methods: To generate optimal PD1-TLR10 fusion proteins, PD1 extracellular domain and TLR10 intracellular domain were connected by transmembrane domain from PD1, CD28, or TLR10, respectively. The fusion protein was co-expressed with second generation anti-CEA CAR in the same retroviral vector. The effector function and the efficacy of fusion protein armored CAR-T cells was evaluated in vitro and in vivo., Results: PD1-TLR10 armored CEA CAR-T cells showed stronger cytotoxicity and cytokine release against CEA-positive tumor cells. Specifically, CAR-T cells with fusion protein containing TLR10 transmembrane domain demonstrated better anti-tumor activity in xenograft mouse model., Conclusion: Our study demonstrated that CEA CAR-T armored with rational designed PD1-TLR10 fusion protein had improved efficacy in colon cancer., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are employees of TriArm Therapeutics and are among the inventors of patent application related to fusion proteins., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies., Competing Interests: Competing interests statement:W.S. and F.K. are co-founders and scientific advisory board members of CastleVax. F.K. has consulted for Merck, Curevac, Seqirus, GlaxoSmithKline (GSK) and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to influenza virus vaccines, which list P.P. and F.K. as co-inventors. The Icahn School of Medicine at Mount Sinai has also filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, and influenza virus therapeutics which list F.K. as co-inventor. W.S. is listed as co-inventor of NDV-based SARS-CoV-2 vaccines.

Proteolysis-Targeting Chimeras (PROTAC) are a bifunctional molecule that binds to a protein of interest (POI) and a ubiquitin ligase, thereby inducing the ubiquitination and degradation of POI. Many PROTACs currently utilize a limited number of ubiquitin ligases, such as von Hippel-Lindau (VHL) and Cereblon. Because these ubiquitin ligases are widely expressed in normal tissues, unexpected side effects can occur. Therefore, to expand the repertoire of ubiquitin ligases that can be utilized in PROTACs, we aimed to develop a versatile system to identify suitable novel ubiquitin ligases for PROTAC-mediated protein degradation using existing PROTACs. Chimeric ubiquitin ligases are constructed by fusing VHL with the ubiquitin ligase of interest that is stably expressed in cells. An existing PROTAC that binds to VHL was added to the cells, and the POI degradation activity was evaluated. In this study, we showed that epidermal growth factor receptor can be degraded by an existing PROTAC utilizing a chimeric ubiquitin ligase that fuses VHL and endoplasmic reticulum-localized ubiquitin ligase, HRD1. These results demonstrate that this novel approach can be used to identify suitable ubiquitin ligases for PROTAC-mediated degradation using existing PROTACs. Expanding the repertoire of ubiquitin ligases that can be utilized for PROTAC by using this versatile system is expected to enable the development of more effective and specific PROTACs for cancer and other diseases., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Human papillomavirus (HPV) is the leading cause of cervical cancer worldwide. The pathogenesis of HPV is mainly dependent on its E7 and E6 proteins. Up to now, different adjuvants have been used to enhance the efficacy of the immune response against these two proteins. In this study, Flagellin (FLA) was used as adjuvant to test adjuvant activity and also see whether its orientation of attachment can affect the immune response pattern. The E7d-FLA and FLA-E7d in pET28a vector were constructed and then the recombinant proteins were expressed in E. coli BL21 (DE3) bacteria under IPTG induction. The expression of recombinant E7d-FLA and FLA-E7d proteins is confirmed by SDS-PAGE and western blot. Then, recombinant fusion proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. The recombinant proteins were checked for endotoxin contamination and then quantified by Bradford. Eight-to-ten-week-old male Balb/C mice were immunized subcutaneously with 10 µg recombinant E7d-FLA, FLA-E7d and HPV16E7d vaccine on days 0, 14 and 28. In addition, PBS and FLA groups were considered as control group. Then, spleen cells were harvested to assess lymphocyte proliferation and IFN-γ, IL-4 and IL-17 cytokines. In addition, mice sera were used for specific total IgG and IgG1, IgG2a, IgG2b and IgM antibodies assessment by ELISA. The results show that E7d-FLA is more potent in the induction of lymphocyte proliferation, CTL response and specific total IgG, IgG2a and IgG2b response, while the FLA-E7d vaccine was associated with more IFN-γ, and IL-17 cytokine response. The results of this study proved the ability of FLA as an adjuvant in fusion with E7d in the induction of cellular and humoral immune responses. In addition, it also emphasizes that antigen-adjuvant orientation can affect the immune response strength and polarization against HPV E7d vaccine candidate. HIGHLIGHTS: Flagellin is attached to HPV-16 E7d at the C- or N-terminus to create E7d-FLA and FLA-E7d candidate vaccines. The E7d-FLA vaccine showed a significant increase in lymphocyte proliferation, CTL response and IgG response versus FLA-E7d vaccine. The FLA-E7d vaccine is associated with a significant increase in IFN-γ and IL-17 cytokines response versus E7d-FLA vaccine. It seems that that antigen-adjuvant orientation is an important parameter in the strength and polarization of immune response in HPV E7d vaccine candidate., Competing Interests: Compliance with ethical standards: This article does not contain any human clinical trial results. The present work contains animal study and it was approved by the Ethics committee of Pasteur Institute of Iran and all experiments were performed according to the Animal Care and use Protocol of Pasteur Institute of Iran. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fc-fusion peptides, also known as peptibodies, are a promising new category of targeted therapeutics that offer alternatives to monoclonal antibodies (mAbs) for cancer treatment. This study focuses on an Fc-fusion peptide consisting of the Fc region of IgG1 and an epidermal growth factor receptor (EGFR)-targeting peptide, GE11, which was identified using the phage display method and demonstrated high affinity for the receptor. The fusion peptide (FcIgG-GE11) was successfully expressed in Escherichia coli and purified using ion-exchange chromatography. Flow cytometry confirmed its specific binding to EGFR. Like Cetuximab, the FcIgG-GE11 peptibody exhibited effective, dose- and time-dependent growth inhibition of EGFR-overexpressing cancer cell lines. Additionally, the results showed that the FcIgG-GE11 peptibody induced cell death or cycle arrest in certain cancer cell lines, with varying responses depending on the cancer type. The results of In-Cell ELISA when comparing the effects of the FcIgG-GE11 peptibody to Cetuximab on Tyr 1173 phosphorylation were similar. In addition, the relative potency of the FcIgG-GE11 peptibody compared to Cetuximab was assessed using the MTT results by Slope Ratio Analysis. These findings suggest that FcIgG-GE11 peptibody can provide a specific and efficient tool for both targeting and treating cancer cells., Competing Interests: Declarations. Conflict of interest: The authors declare that there are no commercial or financial conflicts of interest. Ethics statement: All experiments and procedures were approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1401.023) and performed in accordance with the approved guidelines and regulations., (© 2025. The Author(s).)

Maltose-binding protein (MBP) and glutathione S-transferase (GST) are widely used solubility-enhancing protein tags, typically employed to address various issues related to protein expression and purification. The detection of these tags are usually achieved through binding of corresponding antibodies. Designing low-cost binders as alternatives to antibodies is of great significance. This study employed a de novo design approach, starting with a large number of protein scaffolds and screening out 6 candidate binders targeting MBP and 4 candidate binders targeting GST based on scoring functions. Flow cytometry low-affinity selection and biolayer interferometry (BLI) quantitative results showed that MBP and GST can interact strongly with one or several binders, exhibiting nanomolar binding. Among them, LZMB3 has a binding dissociation constant (K D ) of 54.05 ± 1.46 nM, while LJGB3 and LJGB4 have K D values of 105.4 ± 1.812 nM and 437.9 ± 17.69 nM, respectively., Competing Interests: Declaration of competing interest The work described has not been submitted elsewhere for publication, and all the authors listed have approved the manuscript. There are no conflicts of interest associated with this work., (Copyright © 2025. Published by Elsevier Inc.)

Immune checkpoint inhibitors (ICIs) have significantly advanced the field of cancer immunotherapy. However, clinical data has shown that many patients have a low response rate or even resistance to immune checkpoint inhibitor alone. The underlying reasons for its poor efficacy include the deficiency of immune infiltration and effective CD28/CD80 costimulatory signal in tumor. Discoidin domain receptor 1 (DDR1) has been reported to be negatively related to immune cell infiltration in tumors. Herein, we constructed a soluble fusion protein using CD80, the natural ligand of CD28, in combination with DDR1 inhibitor. Our results demonstrated that CD80-Fc effectively activated T cells and inhibited tumor growth in vivo, even in tumors with poor efficacy of ICIs. Importantly, CD80-Fc fusion protein had a milder affinity against the targets which suggested a potential higher safety than CD28 agonists. Further, in order to promote tumor immune infiltration, we attempted to combine CD80-Fc fusion protein with DDR1 inhibitor for treatment. Our results indicated that using CD80-Fc fusion protein along with DDR1 inhibitor significantly promoted T cell infiltration in tumor microenvironment and more strongly inhibited tumor growth. Therefore, the combination use of CD80 fusion protein and DDR1 inhibitor could become an effective tumor immunotherapy strategy, potentially benefiting a larger number of patients. KEY POINTS: • We successfully constructed, expressed, and purified the recombinant CD80-Fc fusion protein • We demonstrated that CD80-Fc fusion protein has good safety and anti-tumor activity • We demonstrated that using CD80-Fc fusion protein along with DDR1 inhibitor can significantly promote immune infiltration of T cells in tumor microenvironment and more strongly inhibit tumor growth., Competing Interests: Declarations. Ethics approval: The animal experiments were conducted according to guidelines evaluated and approved by the Institutional Animal Care and Use Committee (IACUC), School of Pharmacy, Fudan University (Shanghai, China). We declare that current research is in full compliance with ethical standards. Consent for publication: All authors listed on this manuscript have read and agreed to the publication of this research. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel's voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in the degradation of the low-density lipoprotein receptor (LDLR), and PCSK9 inhibition emerges as an attractive strategy for atherosclerosis management. In this study, extracellular vesicles (EVs) were engineered to nanosponges, which could efficiently adsorb and deliver PCSK9 into lysosomes for degradation. Briefly, nanosponges were engineered by modifying EVs with EGF-A/PTGFRN fusion protein (PCSK9 binding domain EGF-A from the mutant LDLR with higher affinity was fused to the C terminus of prostaglandin F2 receptor negative regulator). The modification endowed the EVs with hundreds of EGF-As displayed on the surface, and thus the capacity to adsorb PCSK9 efficiently. The adsorbed PCSK9 would thus be delivered into lysosomes for degradation when the nanosponges were endocytosed by liver cells, thus releasing endogenous LDLR from degradation. In the ApoE -/- mouse model, tail vein-injected nanosponges were able to degrade PCSK9, increase LDLR expression, lower the LDL-C level, and thus alleviate atherosclerosis. In summary, here we not only develop a novel strategy for PCSK9 inhibition but we also propose a universal method for adsorption and degradation of circulating proteins for disease management., Competing Interests: Declaration of interests L.Y., G.Y., C.W., and M.W. are inventors on patents applied for by Fourth Military Medical University related to PCSK9 inhibition., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

HRas is a small GTPase that plays physiologically important roles in various intracellular signal transduction processes, such as cell growth and proliferation. The structure and action mechanisms of HRas have been well characterized, leading to its widespread use as a molecular switch in bionanomachines. Calmodulin (CaM), a calcium ion-binding protein, acts as an ion-binding molecular switch and activates the target enzymes. We previously demonstrated that the fusion protein of HRas (M13-HRas) with the CaM target peptide M13 at the N-terminus of HRas exhibits reversible regulation of GTPase activity and the interaction between M13-HRas and the downstream signalling factor Raf by calcium ions with CaM. In this study, we prepared two new HRas fusion proteins with the M13 peptide at the C-terminus (HRas-M13) and both termini (M13-HRas-M13) of HRas and analysed the calcium-dependent regulation of HRas function. M13-HRas-M13 more efficiently controlled GTPase, interaction with Raf and the HRas regulator GEF by calcium ions with CaM., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Na v 1.7 is a eukaryotic voltage-dependent Na channel (Na v ) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I-IV). It is involved in pain perception, and VSDs that differ significantly by Na v subtype are targeted in the development of Na v 1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na v 1.7 inhibitors, the chimera protein (Na v Ab/1.7VSD) of Na v from prokaryotic Arcobacter butzleri (Na v Ab), in which extracellular loops of VSD were replaced with homologous loops from Na v 1.7, serves as an effective model. This is because Na v Ab harbors only one VSD and the biological activity of Na v Ab/1.7VSD was previously confirmed. To date, Na v Ab/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of Na v Ab/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na v 1.7-specific peptide inhibitor. The results indicate that Na v Ab/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Na v 1.7 inhibitors using the PERISS screening method., Competing Interests: Declaration of competing interest T. Yamaguchi, T. Okada, and T. Kimura are employees of Veneno Technologies Co., Ltd., which provides the PERISS screening services. The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Paeniclostridium sordellii is responsible for severe infections in horses, cattle, and sheep, however, conventional vaccines exhibit limitations in production and immunogenicity. This study assessed the immunogenicity of a recombinant bacterin composed of a chimera (rQTcsHL) that combines segments from the lethal (TcsL) and hemorrhagic (TcsH) toxins in mice and sheep. Both immunized animal groups exhibited elevated levels of IgG, with the mice demonstrating moderate protection (<50 %) against lethal challenges, comparable to that of the conventional vaccine. Further molecular optimization is essential to enhance its efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

One of the main bottlenecks for recombinant peptide production is choosing the proper cleavage method to remove fusion protein tags from target peptides. While these tags are crucial for inhibiting the activity of the target peptide during heterologous expression, incorporating a cleavage site is essential for their later removal, ensuring the pure sequencing of the peptide. This review evaluates different cleavage methods, including protease-mediated, self-cleavable protein, and chemical-mediated sites, regarding their advantages and limitations. For instance, intein, N pro EDDIE, enterokinase, factor Xa, SUMO, and CNBr are options for residue-free cleavage. Although protease-mediated cleavage is widely used, it can be expensive, due to its own cost added to the whole process. As an alternative, self-cleavable sites eliminate the requirement for proteinases. Another crucial step in defining the proper cleavage method is cost consideration, which relates to the purpose of peptide production. Here, we explore a range of cleavage approaches, meeting the needs of both cost-constrained applications and a more flexible budget. Overall, selecting the most suitable cleavage method should be based on careful consideration of toxicity, cost, accuracy, and specific application requirements to ensure a state-of-the-art approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca 2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Heparinases, including heparinases I-III (HepI, HepII, and HepIII, respectively), are important tools for producing low-molecular-weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK) 5 (R) and a flexible linker (GGGGS) 5 (F) to fuse maltose-binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL-c2X. Compared with their parental fusion protein, MBP-fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK) 5 or (GGGGS) 5 all displayed enhanced thermostability (half-lives at 30°C: 242%-464%). MBP-fused HepIs and HepIIs exhibited higher specific activity (127%-324%), whereas MBP-fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP-fused HepIIs showed a significantly decreased affinity toward heparin with increased K m values (397%-480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP-fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP-fused heparinases with suitable linker designs and their potential for the bioproduction of low-molecular-weight heparin., (© 2024 International Union of Biochemistry and Molecular Biology, Inc.)

The Pichia pastoris expression system is a favorable platform for production of pharmaceutical proteins. Treatment of strains with N-acetyl-L-cysteine (NAC) has been shown to enhance the yield of recombinant proteins, thereby contributing to a reduction in production costs. However, the specific mechanism of action of NAC remains unclear. Previous research has indicated that glutathione (GSH) and autophagy are involved in the increased production of human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) by NAC. This study investigated the potential interaction between GSH and autophagy in the production of HSA-pFSHβ. The findings indicated that sulfhydryl-free antioxidants such as melatonin, vitamin C, or vitamin E did not exhibit similar effects to NAC in enhancing HSA-pFSHβ yield. Moreover, NAC was found to enhance HSA-pFSHβ production by modulating GSH metabolism to reduce GSH consumption, increase total GSH levels, as well as glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities. Additionally, inhibition of autophagy through disruption of autophagy scaffolding proteins Atg1 or Atg11 led to an increase in recombinant HSA-pFSHβ production. Furthermore, NAC significantly decreased the phosphorylation of Slt2, and the absence of the SLT2 gene influenced the effect of NAC on HSA-pFSHβ secretion by modulating mitophagy and GSH metabolism. In conclusion, these results suggest a complex interplay between GSH metabolism and autophagy in the regulation of NAC-induced HSA-pFSHβ secretion., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gang Luo reports was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Survivin is an inhibitor of apoptosis, and expressed in a large number of cancers. As Survivin expression is very low in normal tissues, it assumes significance as a prominent target for tumor diagnosis, prognosis and developing anti-cancer therapies. We report development of a novel triple fusion protein for a prospective vaccine against Survivin in targeted cancer immunotherapy. A gene was synthesized by combining the nucleotides encoding human origin Survivin and heat-labile enterotoxin of Escherichia coli (LTB). Further, nucleotides corresponding to single chain variable fragment (scFv) of a monoclonal having affinity for DEC205 receptor present on dendritic cells, were also incorporated into the gene sequence. This complete gene was expressed to a triple fusion recombinant protein using a bacterial expression vector under the control of robust bacteriophage T7 promoter. The recombinant DC Survivin-LTB protein, with a size of approximately 60 kDa, was purified from the inclusion bodies using affinity based Ni-NTA columns. The purified protein was confirmed by the Western blot, and further characterized with circular dichroism, fluorescence spectroscopy and mass spectroscopy. This molecularly adjuvanted Survivin fusion protein designed to deliver to the dendritic cells for better antigen processing, elicited a stronger anti-Survivin immune response compared to Survivin protein alone. It can be an effective vaccine in active and passive immunotherapies for Survivin expressing cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The study investigates the potentials of the rapid crosslinking hydrogel concoction comprising of natural silk fibroin (SF) and recombinant tailorable collagen-like protein with binding domains for wound repair. The formation of dityrosine crosslinks between the tyrosine moieties augments the formation of stable hydrogels, in the presence of the cytocompatible photo-initiator riboflavin and visible light. This uniquely engineered PASCH (Photo-activated silk fibroin and tailor-made collagen-like protein hydrogel) confers the key advantage of improved biological properties over the control hydrogels comprising only of SF. The physico-chemical characterization of the hydrogels with respect to crosslinking, modulus, and thermal stability delineates the ascendancy of PASCH 7:3 over other combinations. Furthermore, the hybrid protein hydrogel proves to be a favorable cellular matrix as it enhances cell adhesion, elongation, growth, and proliferation in vitro. Time-lapse microscopy studies reveal an enhanced wound closure in human endothelial cell monolayer (EA.hy926), while the gene expression studies portray the dynamic interplay of cytokines and growth factors in the wound milieu facilitating the repair and regeneration of cells, sculpted by the proteins. The results demonstrate the improved physical and biological properties of fabricated PASCH, depicting their synergism, and implying their competency for use in tissue engineering applications., (© 2024 Wiley‐VCH GmbH.)

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807 bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31 kDa fusion protein with Histidine tag (His 6 BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His 6 BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His 6 BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids., Competing Interests: Declarations. Conflict of interest: All the authors declare that this is an innovative work that has not been published in other research journal and they have no conflict of interest between them or personal relationship that might alter the research work summarized in the manuscript. All the authors contributed outstandingly to the work. Ethical Approval: Not Applicable. Informed Consent: Not Applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3'-5' exonuclease, and 3'-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a (+) to form the expression vector pET-30a (+) -TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg)., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cytokine interleukin 6 (IL-6) signals via the IL-6 α-receptor (IL-6Rα or IL-6R) in complex with the gp130 β-receptor. Cell type restricted expression of the IL-6R limits the action of IL-6 mainly to hepatocytes and some immune cells. Here, we show that IL-6 also binds to the IL-11 α receptor (IL-11Rα or IL-11R) and induces signaling via IL-11R:gp130 complexes, albeit with a lower affinity compared to IL-11. Antagonistic antibodies directed against IL-11R, but not IL-6R, inhibit IL-6 signaling via IL-11R:gp130 receptor complexes. Notably, IL-11 did not cross-react with IL-6R. IL-11R has also been identified as an alternative α receptor for the CNTF/IL-6-derived chimeric cytokine IC7, which has recently been shown to induce weight loss in mice. Accordingly, the effects of therapeutic monoclonal antibodies against IL-6 or IL-6R, which both block IL-6 signaling, may be slightly different. These findings provide new insights into IL-6 signaling and therefore offer new potential therapeutic intervention options in the future., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Toxoplasma gondii is a protozoan parasite causing toxoplasmosis, a principal concern for public health and livestock industries. Effective vaccination strategies are crucial for controlling toxoplasmosis, particularly in the lamb, which are significant reservoirs of T. gondii. In addition, ovine toxoplasmosis also causes economic losses due to abortions and reproductive complications. In this study, we evaluated two immunization strategies to elucidate the immune protective potential of T. gondi major surface protein SAG1 fused to the plant heat shock proteins 90-kDa (pHsp90) adjuvant against experimental toxoplasmosis in lambs. We performed an oral administration of fresh leaves homogenate infiltrated with a B- and T-cell antigenic epitope-containing surface protein SAG1 (SAG1HC) fused to Arabidopsis thaliana Hsp90 (AtHsp81.2-SAG1HC) (Plant Vaccine) and a subcutaneous administration of recombinant SAG1HC fused to Nicotiana benthamiana Hsp90 (NbHsp90.3-SAG1HC) produced in Escherichia coli (Recombinant Vaccine). Our results showed that only the Recombinant Vaccine significantly increased anti-rSAG1 total IgG values (∼ 4-fold more than the Vehicle and Control groups). In addition, only lambs immunized with the Plant Vaccine showed a significant increase (∼ 3-fold more than the Vehicle and Control groups) in IFN-γ serum levels after the experimental infection (evaluated 8 days post-challenge). On the other hand, we also observed a statistically significant decrease (∼ 80 % less) in histopathological lesions (injury score) in challenged vaccinated lambs compared to challenged but not vaccinated animals (Vehicle and Control groups). Previously, we showed that the chimera recombinant Gra4-Gra7 protein is an acute marker of human infection. Since Gra4-Gra7 is not connected to the SAG1 immunogen, this chimera allows us to monitor infection in challenged lambs early. All lambs from the Control and Vehicle groups showed higher rates of serological reactivity than lambs from the vaccinated groups, concurrently with increased severity of lesions. These results suggest that the Plant-based and Recombinant Vaccines are promising candidates for controlling T. gondii infection in lambs, with potential benefits for enhancing public health and animal welfare., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2025. Published by Elsevier B.V.)

The novel heavy-chain antibody known as immunoglobulin new antigen receptor (IgNAR) is derived from cartilaginous fishes such as sharks. IgNAR, which binds to antigens with the high specificity and affinity of a conventional IgG antibody and exhibits high resistance to denaturation, has potential as a next-generation antibody in biopharmaceutical and biotechnological applications. High-level expression of recombinant IgNAR in animal cells has been challenging. In our previous study, IgNAR was expressed as a fusion protein with a human IgG Fc region (IgNAR-Fc) in Chinese hamster ovary (CHO) cells, but did not meet the production level required for further research and application. In this study, we sought to identify the production bottleneck in CHO cells as a first step toward achieving abundant production of IgNAR. Using an established IgG high-production CHO cell line as a comparator, we found that the amounts of intracellular dimeric IgNAR-Fc produced in CHO cells were similar to those of intracellular dimeric IgG. Furthermore, the majority of intracellular IgNAR-Fc was retained in the endoplasmic reticulum (ER) and strongly colocalized to ERGIC-53, the cargo receptor for coat protein complex II (COP II)-coated vesicles. These findings suggest that COP II-mediated ER-to-Golgi transport may represent a bottleneck for IgNAR-Fc production in the CHO cell expression system., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Objective: Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level., Methods: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging., Results: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function., Conclusions: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons., Competing Interests: Declaration of competing interest The authors declare no competing interests. The present work was performed in (partial) fulfillment of the requirements for obtaining the degree “Dr. med.” at Friedrich-Alexander-Universität Erlangen-Nürnberg., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mussel foot proteins (mfps) play important roles in surface interaction and underwater adhesion. However, limited production and the lack of adhesion of recombinant mfps have restricted their widespread use. Here, we present a general strategy for enhancing both the expression and function of mfps by connecting multiple protein fragments. We demonstrate that fp-535, the fusion protein comprising fp-5 at each fp-3 terminus, exhibits best adhesion strength up to 1.43 MPa and coating ability, while achieving a higher yield of 91.13 mg/L in the flask culture. After optimizing the fermentation parameters such as temperature and volume as well as the culture medium, the yield of fp-535 was increased to 1.70 g/L in a 5-L bioreactor. Our approach highlights the advantage of fusion mfps and provides potential for the sustainable supply of practical bioadhesive materials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Difficulties exist in obtaining full-length, correctly folded, and soluble papain or papain-like proteases that necessitate the exploration of alternative strategies. This study describes the development of an Escherichia coli strain capable of producing soluble papain without the need for complex and time-consuming in vitro refolding steps. To enhance the production of soluble papain, engineered T7 promoters and a recombinant papain translationally fused with varying tags were constructed. The tags investigated include the maltose-binding protein, small ubiquitin modifier protein, and glutathione transferase. An E. coli SHuffle strain was engineered to accumulate hydrogen peroxide (H 2 O 2 ) by disruption of the redox pathway. This was accomplished by co-expression of the fusion constructs with two human endoplasmic reticulum-resident proteins, thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI). The oxidizing capacity of H 2 O 2 was used to improve disulfide bond formation in papain. The GPx7-PDI fusion dyad played a significant role in consuming harmful H 2 O 2 generated by the SHuffle cells. This consumption of H 2 O 2 helped provide the necessary oxidizing conditions for the efficient production of soluble papain. In shake-flask experiments, the recombinant strain produced ~110 mg/L of papain. Moreover, in batch fermentation, the volumetric yield reached ~349 mg/L. This work provides insights into recombinant papain microbial production that can lead to an industrial viable production strain., Importance: Papain, a cysteine-like protease, has extensive applications across various industries including food, chemical, pharmaceutical, drug, and polymer. However, the traditional isolation of papain from Carica papaya plants results in a complex mixture of proteases. Such protease mixtures result in an inability to understand which component enzyme contributed to substrate conversions. Concentrations of constituent enzymes likely differ based on the ripeness of the papaya fruit. Also, constituent enzymes from papaya differ in optimal activity as a function of temperature and pH. Thus, by using papain-like enzymes from papaya fruit, valuable information on component enzyme activity and specificity is lost. Numerous methods have been reported to purify papain and papain-like enzymes from the crude mixture. Often, methods involve at least three steps including column chromatography to separate five cysteine proteases. Such procedures represent tedious processes to manufacture the pure enzymes in Carica papaya extracts. The numerous uses of papain for industrial processes, as well as the probability that certain components of papain crude mixtures will be preferred for specific applications, necessitate alternative methods such as recombinant expression from microbial production systems to meet the high world demand for papain., Competing Interests: The authors declare no conflict of interest.

In this article we discuss characteristics of fusion protein-based SARS-CoV-2 vaccines. We focus on recombinant vaccine antigens comprising fusion proteins consisting of combinations of SARS-CoV-2-derived antigens or peptides or combinations of SARS-CoV-2 antigens/peptides with SARS-CoV-2-unrelated proteins/peptides. These fusion proteins are made to increase the immunogenicity of the vaccine antigens and/or to enable special targeting of the immune system. The protein-based vaccine approach is exemplified solely in a proof of concept study by using W-PreS-O, a chimeric vaccine based on a single fusion protein (W-PreS-O), combining RBDs from Wuhan hu-1 wild-type and Omicron BA.1 with the hepatitis B virus (HBV)-derived PreS surface antigen adsorbed to aluminum hydroxide. The W-PreS-O vaccine was evaluated in Syrian hamsters which were immunized three times at three-week intervals with W-PreS-O or with aluminum hydroxide (placebo) before they were infected with Omicron BA.1. Neutralizing antibody (nAB) titers, weight, lung symptoms, and viral loads, as measured using RT-PCR in the upper and lower respiratory tracts, were determined. In addition, infectious virus titers from the lungs were measured using a plaque-forming assay. We found that W-PreS-O-vaccinated hamsters developed robust nABs against Omicron BA.1, showed almost no development of pneumonia, and had significantly reduced infectious virus titers in the lungs. Importantly, the viral loads in the nasal cavities of W-PreS-O-vaccinated hamsters were close to or above the PCR cycle threshold considered to be non-infectious. The data of our proof-of-concept study provides compelling evidence that the W-PreS-O vaccine has protective effect against Omicron BA.1 in a Syrian hamster in vivo infection model and thus support the promising results obtained also for other fusion protein-based SARS-CoV-2 vaccines., Competing Interests: RV has received research grants from HVD Life-Sciences, Vienna, Austria, WORG Pharmaceuticals, Hangzhou, China and from Viravaxx AG, Vienna, Austria. He serves as consultant for Viravaxx AG. RV and PG are authors on a patent application regarding the vaccine. The authors with Russian affiliation declare that they have prepared the article in their “personal capacity” and/or that they are employed at an academic/research institution where research or education is the primary function of the entity. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Gattinger, Kozlovskaya, Lunin, Gancharova, Sirazova, Apolokhov, Chekina, Gordeychuk, Karaulov, Valenta and Ishmukhametov.)

Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 h. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 µg/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation (t < 0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Lectins are produced in almost all life forms, can interact with targets (glycans) in a cross-kingdom manner and have served as valuable tools for studying glycobiology. Previously, a bacterial lectin, named Streptomyces hemagglutinin (SHA), was found to agglutinate human type B erythrocytes. However, the binding of SHA to mammalian cell types other than human erythrocytes has not been explored. To address this, we produced a recombinant fusion protein, with the mCherry reporter protein proceeding the SHA protein (referred to as mCherry-SHA), and performed co-immunofluorescence staining analysis. We focused on the normal pancreas in this study because glycans on pancreatic cells have been associated with initiation and progression of pancreatic cancer, a deadly disease. We found that only acinar, but not ductal or endocrine cells were stained positively with mCherry-SHA from embryonic day (E) 18.5 to 35 weeks old mice; in contrast, E12.5 and E15.5 pancreas display minimal mCherry-SHA binding. In adult humans, mCherry-SHA also targeted acinar cells specifically; however, only tissue from blood type B donors, but not type A or O donors, showed positivity. Together, these results demonstrate that SHA can bind to normal murine and human pancreatic acinar cells and that SHA-binding glycans are developmentally regulated., Competing Interests: Declarations. Competing interests: M.K., Y.F-Y, and K.B. declare holder for United States Patent No. US11,767,351 B2. Date of Patent: Sept. 26, 2023. Other authors declare no conflicts of interest with the contents of this article., (© 2025. The Author(s).)

HUG is the HELP-UnaG recombinant fusion protein featuring the typical functions of both HELP and UnaG. In HUG, the HELP domain is a thermoresponsive human elastin-like polypeptide. It forms a shield enwrapping the UnaG domain that emits bilirubin-dependent fluorescence. Here, we recapitulate the technological development of this bifunctional synthetic protein from the theoretical background of its distinct protein moieties to the detailed characterization of its macromolecular and functional properties. These pieces of knowledge are the foundations for HUG production and application in the fluorometric analysis of bilirubin and its congeners, biliverdin and bilirubin glucuronide. These bile pigments are metabolites that arise from the catabolism of heme, the prosthetic group of cytochromes, hemoglobin and several other intracellular enzymes engaged in electron transfer, oxygen transport and protection against oxygen free radicals. The HUG assay is a powerful, user-friendly and affordable analytical tool that alone supports research at each level of complexity or taxonomy of living entities, from enzymology, cell biology and pathophysiology to veterinary and clinical sciences.

In this study, we designed a fusion protein, rhCR, by combining human collagen with the self-assembling peptide RADA-16 using genetic engineering technology. The rhCR protein was successfully expressed in Pichia pastoris . The rhCR can self-assemble into a three-dimensional nanofiber network under physiological conditions. The lyophilized rhCR sponge exhibited high elasticity modulus and stable swelling properties. In vitro experiments confirmed that the rhCR had good biocompatibility and could significantly promote the adhesion, proliferation, and migration of fibroblasts (L929), upregulating the expression of genes such as Vim , Fgf , Vegf , and Tgf-β3 in L929 cells. When applied to a mouse liver hemorrhage model, rhCR hemostatic sponges rapidly formed nanofibers on the ruptured liver surface, activated platelet CD62P, and significantly reduced blood loss and bleeding duration compared to the recombinant human collagen (rhCol) alone. Furthermore, the rhCR application markedly accelerated wound healing in a mouse full-thickness skin defect model, with the wound healing rate in the rhCR group being 2.6 times that of the untreated group and 1.7 times that of the rhCol group on day 6 postinjury. Histological and immunofluorescence analyses revealed that the rhCR promoted collagen deposition and epidermal regeneration and improved the quality of skin tissue repair by stimulating tissue cells to produce cytokines, growth factors, and immune factors through immunological regulation. The rhCR fusion protein combines the advantages of collagen and RADA-16, overcoming the limitations of their separate use in hemostatic and tissue engineering applications. This biomaterial and its design idea hold promise for a variety of regenerative applications.

The re-emergence of the mpox pandemic poses considerable challenges to human health and societal development. There is an urgent need for effective prevention and treatment strategies against the mpox virus (MPXV). In this study, we focused on the A35R protein and created a chimeric A35R-Fc protein by fusing the Fc region of IgG to its C-terminal. We then assessed its reactivity with A35R-specific antibodies and human convalescent plasma, as well as its immunogenicity. Our findings indicate that the A35R-Fc protein significantly enhances affinity to A35R antibodies compared to the commercially available A35R protein and exhibits considerable reactivity to human plasma. Additionally, mice immunized with A35R-Fc exhibited increased neutralizing antibody titers against the live MPXV. These results support the potential of Fc domain chimeric antigens as a strategy to enhance the efficacy of subunit vaccines targeting the MPXV.

One of the most significant bacteriophage technologies is phage display, in which heterologous peptides are exhibited on the virion surface. This work describes the display of λ decorative protein D λ linked to the E protein domain III of Zika virus (D λ -ZE DIII ), to the GFP protein (D λ -GFP), or to different domain III epitopes of the E ZIKV protein (D λ -TD), exhibited on the surface of an in vitro evolved lambda phage (λ evo ). This phage harbors a gene D deletion and was subjected to directed evolution using Escherichia coli W3110/pD λ -ZE DIII as background. After 20 days (20 cycles of dilution), the λ evo phage developed a ~ 22% genome deletion affecting the non-essential λ b region, rendering a more stable phage that exhibited fusion proteins D λ -ZE DIII or D λ -GFP but not D λ -TD. Despite the λ evo system was able to decorate itself with the D λ -ZE DIII protein, the production of viral particles was ~ 1000-fold lower than the λ wild-type, due to the unexpected D λ -ZE DIII protein aggregation into bacterial inclusion bodies. Decorated phages (10 6 PFU (plaque forming units)/100 µl) were inoculated into BALB/c mice, and subsequent dot blot and Western blot immunoassays proved the production of murine antibodies against ZIKV (Zika virus). This multipurpose λ evo phage display platform may be used interchangeably with other more soluble peptides, providing better yields. KEY POINTS: • λ evo  platform for displaying recombinant peptides. • Directed evolution to generate λ evo  with more efficient decoration. • Antigenic reaction in BALB/c mice by inoculating λ evo  with recombinant peptides., Competing Interests: Declarations. Ethics approval: Experiments were performed following IACUC guidelines. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

Keratinases are valuable enzymes for converting feather keratin waste into bioactive products but often suffer from poor substrate specificity and low catalytic efficiency. This study reported the creating of a novel keratinase with targeted adherence and specific degradation on feather keratins by fusing prepeptidase C-Terminal (PPC) domain. A PPC domain of metalloprotease E423 specifically adsorbed feather keratins by hydrogen bonds and hydrophobic interactions in a time- and temperature-dependent manner. Stepwise N-/C-terminal truncations disclosed the essential core sequence composed of 21 amino acid residues determining the keratin-targeted adherence. Fusion of the core fragment with a flexible linker (GGGGS) 1 achieved the optimal secretion, and improved the catalytic efficiency of a representative keratinase 4-3 Ker -MAV by 0.97-fold. Moreover, the feather degradation rate increased from 65 to 82%, representing the highest reported performance for a keratinase. This PPC-fusion strategy opens new horizons in enzyme engineering, promising not only to revolutionize keratin waste valorization but also to inspire the design of substrate-specific biocatalysts across diverse industrial applications.

Sensory capabilities are crucial for cells to interact with their environment. To mimic these functions in synthetic cells, we developed sensory globular protein vesicles (GPVs) made entirely of recombinant fusion proteins through self-assembly under aqueous conditions. GPVs demonstrate sensory functions via the formation of the FKBP-FRB ternary complex with the signaling molecule, rapamycin. The sensory domain of FKBP or FRB was genetically fused to a fluorescent protein and leucine zipper, which self-assemble into vesicles by forming amphiphilic building blocks through high-affinity binding to a counter leucine zipper fused to an elastin-like polypeptide (ELP) above its lower critical solution temperature. We observed intervesicle aggregation in a time- and concentration-dependent manner upon rapamycin binding, confirmed by colocalization studies and statistical analysis. This system enhances our understanding of protein vesicle functionality for sensing and offers a basis for exploring GPVs as models to replicate key cellular processes in synthetic cells.

Pompe disease is a tissue glycogen disorder caused by genetic insufficiency of the GAA enzyme. GAA enzyme replacement therapies for Pompe disease have been limited by poor lysosomal trafficking of the recombinant GAA molecule through the native mannose-6-phosphate-mediated pathway. Here, we describe the successful rational engineering of a chimeric GAA enzyme that utilizes the binding affinity of a modified IGF-II moiety to its native receptor to bypass the mannose-6-phosphate-mediated lysosomal trafficking pathway, conferring a significant increase in cellular uptake of the GAA enzyme. We also demonstrate the ablation of binding between our modified IGF-II tag and two off-target receptors: IGF-I receptor and insulin receptor, as well as preserved enzymatic activity of the chimeric GAA molecule., (© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on the surface of activated T cells. Upon interaction with its cognate ligand, OX40L, OX40 transmits costimulatory signals to antigen-primed T cells, promoting their activation, differentiation, and survival - processes essential for the establishment of adaptive immunity. Although the OX40-OX40L interaction has been extensively studied in the context of disease treatment, developing a substitute for the naturally expressed membrane-bound OX40L, particularly a multimerized OX40L trimers, that effectively regulates OX40-driven T cell responses remains a significant challenge. In this study, we successfully engineered soluble OX40L-fusion proteins capable of robustly activating OX40 on T cells. This was achieved by incorporating functional multimerization domains into the TNF homology domain of OX40L. These OX40L proteins bound to OX40, subsequently activated NF-κB signaling, and induced cytokine production by T cells in vitro . In vivo , mice treated with one of the OX40L-fusion proteins - comprising a single-chain OX40L trimer linked to the C-terminus of the human IgG1 Fc domain, forming a dimer of trimers - exhibited significantly enhanced clonal expansion of antigen-specific CD4 + T cells during the primary phase of the immune response. A comparable antibody-fusion single-chain TNF protein incorporating 4-1BBL, CD70 (CD27L), or GITRL in place of OX40L elicited similar in vivo T cell responses. Thus, we propose that optimizing the multimerization of OX40L proteins through innovative design strategies may facilitate the development of more effective agonists for targeted immunotherapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be consulted as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Sato, Nagai, Suzuki, Ito, Matsuyama, Shibui, Morita, Hikosaka-Kuniishi, Ishii and So.)

Introduction: Brucellosis is a widespread zoonotic disease that poses a considerable challenge to global public health. Existing diagnostic methods for this condition, such as serological assays and bacterial culture, encounter difficulties due to their limited specificity and high operational complexity. Therefore, there is an urgent need for the development of enhanced diagnostic approaches for brucellosis., Methods: Tandem mass tag (TMT) proteomic analysis was conducted on the wild-type strain Brucella abortus ( B. abortus ) DT21 and the vaccine strain B. abortus A19 to identify proteins with high expression levels. The proteins that exhibited high expression in the wild-type strain were selected based on the proteomic results. Subsequently, B-cell linear epitopes were predicted using multiple computational tools, including ABCpred, SVMTriP, BCPred, and Bepipred Linear Epitope Prediction 2.0. These epitopes were concatenated to construct a multiepitope fusion protein. Following prokaryotic expression and purification, an indirect enzyme-linked immunosorbent assay (iELISA) was developed. A total of 100 positive serum samples, 96 negative serum samples, and 40 serum samples from patients infected with other pathogens were collected and analyzed using the established iELISA. Furthermore, the protein was assessed for its capability to differentiate human brucellosis from lipopolysaccharide (LPS)., Results: Proteomic analysis revealed the presence of 152 proteins with high expression levels in the wild-type strains. A multiepitope fusion protein, comprising a total of 32 predicted B-cell linear epitopes, was successfully prepared. The results from the iELISA indicated that the multiepitope fusion protein exhibited exceptional diagnostic performance, evidenced by an area under the receiver operating characteristic curve (AUC) of 0.9576, a sensitivity of 0.9300, and a specificity of 0.8542. In comparison to the commonly utilized LPS antigen, the fusion protein demonstrated a reduced level of cross-reactivity., Conclusions: A novel multiepitope fusion protein has been successfully developed utilizing bioinformatics and TMT proteomics technology. This fusion protein demonstrates significant potential as a diagnostic antigen for brucellosis, exhibiting high sensitivity and specificity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Wu, Yuan, Guo, Xie, Yao and Yin.)

SARS-CoV-2 has continued spreading around the world in recent years since the initial outbreak in 2019, frequently developing into new variants with greater human infectious capacity. SARS-CoV-2 and its mutants use the angiotensin-converting enzyme 2 (ACE2) as a cellular entry receptor, which has triggered several therapeutic strategies against COVID-19 relying on the use of ACE2 recombinant proteins as decoy receptors. In this work, we propose an ACE2 silent Fc fusion protein (ACE2-hFcLALA) as a candidate therapy against COVID-19. This fusion protein was able to block the binding of SARS-CoV-2 RBD to ACE2 receptor as measured by ELISA and flow cytometry inhibition assays. Moreover, we used classical neutralization assays and a progeny neutralization assay to show that the ACE2-hFcLALA fusion protein is capable of neutralizing the authentic virus. Additionally, we found that this fusion protein was more effective in preventing in vitro infection with different variants of interest (alpha, beta, delta, and omicron) compared to the D614G strain. Our results suggest the potential of this molecule to be used in both therapeutic and preventive settings against current and emerging mutants that use ACE2 as a gateway to human cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Bermúdez-Abreut et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents, and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue, in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas, but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here, we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells, and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method, new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule Z DGCR2:AM106 displayed nanomolar affinity, suitable stability and biodistribution, with negligible toxicity to islets, qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells., Competing Interests: Declarations. Competing interests: O.E. is an employee of Antaros Tracer. O.E. and O.K. are minority shareholders of Antaros Tracer AB, which hold patents related to DGCR2 binding peptides. Otherwise, the authors declare no competing interests. Ethics approval and consent to participate: All the performed procedures were in accordance with the ARRIVE guidelines for animal research. The animal experiments were ethically reviewed, authorized by the Animal Ethics Committee of the Swedish Animal Welfare Agency and carried out in accordance with the European Directive 2010/63/EEC and the institutional guidelines at Uppsala University (UFV 2007/724)., (© 2024. The Author(s).)

Saccharomyces boulardii and Saccharomyces cerevisiae share over 99 % genetic similarity yet exhibit distinct metabolic traits. While the cell surface display system of S. cerevisiae is well-documented, the equivalent system in S. boulardii has yet to be fully characterized. This study investigates the cell surface display system of S. boulardii for the expression of a heterologous protein using different anchor proteins. Six strains expressing the enhanced green fluorescent protein (Egfp) and an anchor protein as a fusion protein were constructed to visualize the cell surface display system. Then a heterologous endo-inulinase protein was expressed with selected anchor proteins through fluorescence intensity comparison. Analysis by fluorescence microscopy revealed that the anchor protein Sed1 exhibited the highest fluorescence intensity. Furthermore, expressed selected anchor proteins and heterologous protein, endo-inulinase, the engineered strain could degrade and consume almost inulin in 72 h. Through endo-inulinase expression, we confirmed that not only Egfp but also heterologous protein is well expressed, and we successfully built an S. boulardii cell surface display system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cell division is a fundamental biological process, essential for sustaining life on Earth. Accurate replication followed by uniform segregation of the genome is required to ensure cell division is sustainable and reduces the likelihood of aneuploidy. The cell cycle has various checkpoints to safeguard proper replication, for example, the spindle assembly checkpoint (SAC) which ensures that all chromosomes are correctly aligned and attached to the spindle, before the transition to anaphase. The precise function of the SAC and SAC components in plants is so far unclear. First, the high level of polyploidy in plants raises concerns about the efficacy of the SAC. Second, many plant SAC components are implicated in other cellular processes, such as MAD1, which has been implicated in the reproductive transition of Arabidopsis thaliana. Overexpression of GFP fusions of core SAC components provides a key route to establish the functions of the different SAC components in plants. Here we describe two methods for agrobacterium-mediated transformation of plants., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fusion proteins are valuable molecules to meet different demands related to the development of biopharmaceuticals and bioprocesses. In human therapy, they are used to improve the half-life of biologics by modifying the biophysical properties of the proteins. In biotechnology, the design of fusion proteins can standardize the establishment of production clones and the purification process. Analytical detection capabilities of the fusion partner and binding to affinity ligands play a crucial role. For the generation of the recombinant cell line, the maturation of the protein and the secretion are also crucial factors, which can be significantly influenced by the fusion partner and can determine the final yield of the bioprocess. Here we present a protocol to recombine the human extracellular domain of CD19 with the human serum albumin domain 2 resulting in a fusion protein CD19-AD2 including a flexible linker sequence in the interface between the C-terminus of CD19 and the N-terminus of HSA-D2 and a terminal His12-tag. The two fragments are independently amplified with primers allowing to genetically connect the two fragments in the next step by overlap extension PCR. By this strategy, the linker sequence as well as the albumin fragment can be chosen individually to be flexible in the fine-tuning of the final protein. The amplified product is then cloned into a mammalian expression vector suitable to generate a recombinant transient or stable cell culture. This workflow can be applied to any protein sequence by adapting the specific primer sequences., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Respiratory syncytial virus (RSV) is one of the most important etiologies of acute respiratory infections that cause bronchiolitis in children under 5 years of age. Treatments are expensive, no vaccine is available, and this is an important cause of hospitalization. Costimulatory molecules have been reported to be good inducers of antiviral type 1 immune response. This study aimed to generate a recombinant vaccine with the RSV F protein fused to 4-1BBL to evaluate the activation of an antiviral response in vitro and the production of neutralizing antibodies in vivo . The codon-optimized F gene was subcloned into an expression vector as follows: streptavidin core, gene F, and costimulatorytumor necrosis factor receptor superfamily member 9 -TNFRS9- ligand (4-1BBL). After the induction of expression in Escherichia coli C43, the recombinant protein (SA-F3x-4-1BBL, denominated SF4) was purified and verified by western blotting. Cultured RAW264.7 macrophages were stimulated with SF4 protein, then tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), p38, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) were analyzed by western blot, and mRNA cytokines were analyzed by RT-qPCR. Finally, male C57BL/6 mice were inoculated with SF4, and the generation of anti-RSV neutralizing antibodies and serum cytokines was examined. SF4 had a size of 84.4 kDa with a 5.6% yield. SA-F-4-1BBL upregulated TRAF2, TNF-α, and interferon (IFN)-γ expression levels and activated p38 mitogen-activated protein kinase and NF-κΒ pathways in RAW264.7 cells. Importantly, antibodies capable of neutralizing RSV infection and producing type 1 cytokines were detected in the sera of immunized animals. These results suggest that the fusion protein SF4 activates the 4-1BBL signaling pathway, resulting in an effective antiviral response mediated by neutralizing antibodies and antiviral cytokines.

Expression of GRF3-GIF1 chimera significantly enhanced regeneration and transformation efficiency in soybean, increasing the number of transformable cultivars. Moreover, GmGRF3-GIF1 can be combined with CRISPR/Cas9 for highly effective gene editing., (© 2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Botulinum neurotoxins (BoNTs), including serotypes A and E, are potent biotoxins known to cause human poisoning. In addition to the critical protective antigen found in the full BoNT molecule, the receptor binding domain (Hc domain), BoNTs also harbour another essential protective antigen-the light chain-translocation domain (L-HN domain). Leveraging these pivotal protective antigens, we genetically engineered a series of inactivated chimeric molecules incorporating L-HN and Hc domains of BoNT/A and E. The structure of these chimeric molecules, mirror BoNT/A and E, but are devoid of enzyme activity. Experimental findings demonstrated that a lead candidate mEL-HN-mAHc harnessing the inactivated protease LCHN/E with the mutated gangliosides binding site Hc/A (mE-mA) elicited robust immune protection against BoNT/A and E simultaneously in a mouse model, requiring low immune dosages and minimal immunisations. Moreover, mE-mA exhibited high protective efficacy against BoNT/A and E in guinea pigs and New Zealand white rabbits, resulting in elevated neutralising antibody titres. Furthermore, mE-mA proved to be a more stable and safer vaccine compared to formaldehyde-inactivated toxoid. Our data underscore the genetically engineered mE-mA as a highly effective bivalent vaccine against BoNT/A and E, paving the way for the development of polyvalent vaccines against biotoxins., (© 2024 John Wiley & Sons Ltd.)

Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 μg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites., (© 2024 UICC.)

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Photothermal therapy (PTT) is an innovative cancer treatment that leverages heat generated from near-infrared light exposure to induce tumor cell death. A major challenge in PTT is achieving precise delivery of the photothermal agent to tumor tissues to maximize efficacy and minimize off-target effects. In this study, we introduce a novel ligand-coupled photothermal reagent that addresses this challenge by leveraging the high-affinity HER2 affibody Z HER2:2891 (referred to as Z HER2 ), conjugated with indocyanine green (ICG) for targeted delivery. Polyethylene glycol (PEG) was incorporated as a hydrophilic linker to further optimize photothermal conversion efficiency and enhance tumor-specific targeting. Among the conjugates tested, Z HER2 -PEG 1000 -ICG, modified with a PEG chain of 1000 Da molecular weight, demonstrated exceptional performance. In vitro studies revealed that Z HER2 -PEG 1000 -ICG specifically bound to HER2-expressing cells and effectively induced cell death. In vivo experiments using HER2-positive N87 tumor-bearing mice showed that Z HER2 -PEG 1000 -ICG accumulated highly and specifically in tumor tissues over an extended period. Upon light irradiation, this conjugate caused a significant rise in temperature at the tumor site, resulting in complete tumor elimination with a single photothermal treatment. This PEG-modified affibody-ICG conjugate represents a precise and effective approach to PTT, offering a promising new therapeutic strategy for cancer treatment with the potential to significantly impact future cancer therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)