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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.)

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.)

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.)

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.)

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.)

A 47-year-old woman was diagnosed with myotonic dystrophy when admitted for traumatic subarachnoid hemorrhage. Her glycemic control was poor despite administration of pioglitazone, a PPARɤ agonist, and subcutaneous insulin infusion. However, adding a GLP-1 receptor (GLP-1R) agonist markedly improved blood glucose levels, resulting in eventual insulin withdrawal. Genetic testing revealed a heterozygous variant, p.R131Q, in the GLP1R (rs3765467), a common variant in Asia. This variant is known to be associated with increased endogenous insulin from beta cells in response to exogenous GLP-1 infusion. This is the first report and short review of a Japanese case of myotonic dystrophy accompanied by GLP-1R gene polymorphism., 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 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

One of the promising drug delivery tools is ferritin, which features high stability at a wide range of conditions and protects cargo by its spherical protein shell. We studied the self-assembly into homoglobules of ferritin from H. pylori and a chimeric protein ferritin-SUMO. We exposed the globules to pH-driven dis/reassembly and in both cases we observed two fractions during size exclusion chromatography (SEC) procedure. The higher molecular weight fraction contained fully assembled globules of ferritin and ferritin-SUMO that well coincides with literature. Interestingly, the lower molecular weight fraction contained intermediate subglobular oligomers that also formed globules, but on a time scale of hours, while being under physiological conditions. We performed biochemical characterization of this fraction and found that, in the case of ferritin, it contained almost the whole range of intermediate oligomers with different stoichiometry. In contrast, the ferritin-SUMO fraction contained only two distinct states: dimers and globules, without any other ferritin-SUMO intermediate oligomers. We built AlphaFold-derived schemes of ferritin and ferritin-SUMO self-assembly which also indicated differences in their assembly pathways. Our results could potentially open the possibility of cargo loading into ferritins at physiological conditions and improved purification of ferritin-based products if using a ferritin-SUMO modification with following cleavage of the SUMO-tag by the SUMO protease., 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.)

IFN-λs hold promise as therapeutic candidates against mutable respiratory viruses, but their efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) remains unclear. In this study, we expressed a recombinant fusion protein consisting of porcine ISG15 linked porcine IFN-λ3 (ISG15-IFN-λ3) via a rigid protein linker in Escherichia coli (E. coli). In vitro experiments demonstrated that treatment of porcine alveolar macrophage (PAM)-derived CRL-2843 cells with ISG15-IFN-λ3 induced upregulation of several Interferon-stimulated Genes (ISGs) proteins, including ISG15, ISG56, and HERC5. CRL-2843 cells pretreated with ISG15-IFN-λ3 exhibited heightened resistance to Newcastle disease virus infection, while PRRSV-permissive cells treated with ISG15-IFN-λ3 before and during PRRSV exposure showed significantly inhibited PRRSV replication as well. In animal experiments, at 21 days post-infection, ISG15-IFN-λ3-treated piglets displayed milder lung tissue pathology and significantly reduced serum PRRSV-RNA, indicating enhanced viral clearance and faster recovery. Additionally, PAMs collected from ISG15-IFN-λ3-treated piglets showed significantly reduced mRNA expression of representative cytokines, chemokines, suggesting that ISG15-IFN-λ3 treatment may mitigate pneumonia severity by reducing the levels of these inflammatory mediators. These findings indicate that recombinant ISG15-IFN-λ3 expressed in E. coli may serve as a novel, effective, and affordable agent for treating severe PRRSV infection in piglets, potentially benefiting the pork industry., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Recombinant fusion biotechnology is a powerful tool for producing antimicrobial peptides (AMPs), which can contribute to limiting the number of potentially infectious microorganisms. AMPs are often expressed in fusion with a carrier protein, a strategy that prevents toxic effects on host bacterial cells and protects them from proteolytic degradation. Among the many fusion carriers available, elastin-like polypeptides offer several valuable advantages related to their unique thermo-responsive behavior. The Human Elastin-like Polypeptide was successfully employed to produce a model fusion construct with the indolicidin domain. Recently, an elastin-based variant of this polypeptide was developed, modulating the sequence of the hydrophobic domains, thus enhancing the phase transition properties. Here, a new carrier based on this sequence that was produced, physicochemically characterized, and employed as a fusion partner for the indolicidin is described. This work aims to compare two different elastin-based carriers and their indolicidin fusion derivatives, as well as to determine, based on their properties, which may be the most advantageous carrier for producing antimicrobial domains. This study was focused on the elastin-like polypeptides as a tunable expression platform particularly suitable for producing AMPs and for their integration in biomimetic interfaces that acquire the capacity to inhibit bacterial growth., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Antonella Bandiera, Laura Colomina-Alfaro, Artemis Stamboulis report financial support was provided by MSCA ITN Horizon 2020 grant n. 861138. Antonella Bandiera, Laura Colomina-Alfaro, Angela Ivask, Abeer Shaalan, Lucy Di Silvio, Artemis Stamboulis report financial support was provided by Horizon Europe STOP project, grant No 101057961. Antonella Bandiera, Paola Sist report financial support was provided by PNRR iNEST project, Interconnected Nord-Est Innovation, funded by the EU Recovery fund. Angela Ivask reports financial support was provided by Estonian Research Council projects PRG1496 and by Estonian Ministry of Education and Research (TK210). 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Fusion proteins have the potential to become the new norm for targeted therapeutic treatments. Highly specific payload delivery can be achieved by combining custom targeting moieties, such as V HH domains, with active parts of proteins that have a particular activity not naturally targeted to the intended cells. Conversely, novel drug products may make use of the highly specific targeting properties of naturally occurring proteins and combine them with custom payloads. When designing such a product, there is rarely a known structure for the final construct which makes it difficult to assess molecular behaviour that may ultimately impact therapeutic outcome. Considering the time and cost of expressing a construct, optimising the purification procedure, obtaining sufficient quantities for biophysical characterisation, and performing structural studies in vitro, there is an enormous benefit to conduct in silico studies ahead of wet lab work. By following a repeatable, streamlined, and fast workflow of molecular dynamics assessment, it is possible to eliminate low-performing candidates from costly experimental work. There are, however, many aspects to consider when designing a novel fusion protein and it is crucial not to overlook some elements. In this work, we suggest a set of user-friendly, open-source methods which can be used to screen fusion protein candidates from the sequence alone. We used the light chain and translocation domain of botulinum toxin A (BoNT/A) fused with a selected V HH domain, termed here LC-H N -V HH , as a case study for a general approach to designing, modelling, and simulating fusion proteins. Its behaviour in silico correlated well with initial in vitro work, with SEC HPLC showing multiple protein states in solution and a dynamic protein shifting between these states over time without loss of material., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests to declare. This research was sponsored by Ipsen Bioinnovation. S.M. Liu was an employee of Ipsen at the time of the work & K.A. Bunting is an employee of Ipsen., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Syphilis (T. pallidum) is a systemic and sexually transmitted disease. Over 80 % of syphilis infections are asymptomatic, and some patients tend to conceal high-risk sex histories. Therefore, fast and effective screening of syphilis is crucial to control its spread. In this study, a methodology, combining a luciferase immuno-magnetic separation system (LIMS) with an automated magnetic-processing machine, has been designed to detect syphilis-specific antibodies against TPP17 of T. pallidum. Protein A/G-immobilized magnetic particles captured the antibodies in serum and then were specifically labeled with a chimeric protein of TPP17 with a luciferase hGluc. The magnetic separation was automatically executed. It takes only 25 min from samples to results for 32 samples once. Further, for clinical samples, antibody-negative serum samples from pregnant women were unexpectedly found to have higher bioluminescence than regular sera. Innovatively, two cutoff values were determined. ROC (receiver operating characteristic) analyses showed both the sensitivity and the specificity of 100 % for the regular sera, and the sensitivity and the specificity of 96.5 % and 100 % for the sera during pregnancy. The methodology was 32 times more sensitive than ELISA. The automated platform can potentially be commercialized and would be of great significance in controlling the spread of syphilis., 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.)

Enzymatic PET recycling has emerged as a promising green solution in addition to mechanical recycling, but low soluble expression levels of the inherently hydrophobic PET hydrolases hinder large-scale applications. Here, we propose a novel strategy for enhanced production of FastPETase in Escherichia coli using co-expression of molecular chaperones from Ideonella sakaiensis. Co-expression of cognate DnaK and DnaJ chaperones significantly increased soluble FastPETase expression (up to 2.5-fold), surpassing commercial chaperone plasmids. Furthermore, a combinatorial approach employing co-expression of DnaK/DnaJ chaperones and fusion of FastPETase with the VNp6-tag significantly boosted FastPETase secretion, yielding over 2 g/L of target protein in a 5-l bioreactor. Notably, the crude FastPETase in fermentation broth displayed comparable PET hydrolysis effects to the purified enzyme. This work not only provides new insights into the process of chaperones in protein folding but also suggests a novel and efficient strategy for producing recombinant proteins., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

3-Deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) are key enzymes in the shikimate pathway responsible for aromatic amino acid biosynthesis in bacteria. This study investigated the functional interplay between the DAH7PS and CM domains within the bifunctional enzyme PniDAH7PS-CM from Prevotella nigrescens, a representative of the chimeric enzyme group DAH7PS-CM that is primarily distributed in the Prevotella and Porphyromonas genera. Analysis of the surface polarity demonstrated that DAH7PS and CM domains rely on hetero-domain polar interactions for their catalytic functions, rather than hydrophobic contacts. We evaluated the effects of NaCl on the catalytic activity, conformation, thermal stability, and molecular aggregation of PniDAH7PS-CM at varying NaCl concentrations (0, 150, and 300 mM). Results demonstrated that increasing NaCl concentrations significantly reduced the enzymatic activities of both DAH7PS and CM, with a complete loss of DAH7PS function at 300 mM NaCl. Notably, high NaCl concentrations promoted a more extended conformation of PniDAH7PS-CM and interfere with enzyme aggregation, suggesting that NaCl modulates the inter-domain interactions. Our findings suggest that Na⁺ ions, as kosmotropic agents, likely via enhancing the hydration layer on the enzyme's surface, stabilizes PniDAH7PS-CM structure but disrupting essential polar interactions for catalysis. Conversely, Cl⁻ ions may act as chaotropic agents, further impairing these interactions. This study illuminates the balance between salt ion concentration and enzyme functionality, offering insights for developing therapeutic strategies targeting bacterial metabolism and growth in the context of periodontal diseases., 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 Inc. All rights reserved.)

The substantial accumulation of polyethylene terephthalate (PET) plastic waste in the environment has exacerbated the issue of plastic pollution. The biodegradation of PET plastics using biological enzymes has garnered considerable attention due to its efficiency and environmentally friendly nature. Nevertheless, the low binding affinity of PET plastics presents a significant limitation to the application of biocatalysts in their degradation. This study endeavors to engineer a fusion protein comprising the anchor peptide LCI, derived from Bacillus subtilis A014, and a thermally stabilized variant of Thermobifida fusca cutinase, D204C/E253C (Tfuc2), with the objective of augmenting its polyethylene terephthalate (PET) degradation efficacy. The findings demonstrate that LCI exhibits a high binding affinity for PET, and the hydrolytic efficiency of the LCI-containing fusion protein is enhanced by a factor of 1.8-34.5 compared to the free Tfuc2 enzyme. The enzymatic characteristics and molecular dynamics simulation outcomes indicate that the improved hydrolytic efficiency of PET may originate from the flexible oscillatory behavior of LCI, which exhibits a high binding affinity for PET. This study presents a novel methodology for the enzymatic degradation of PET plastic waste., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600 ). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

TBVs are suggested to inhibit parasite transmission from humans to Anopheles mosquitoes. For the transmission of Plasmodium parasite, a variety of factors are included in gametes fusion phase. In this step, conserved male-specific generative cell specific 1 antigen is necessary for fusion of cytoplasmic membranes of micro- and macro-gametocytes and zygot formation. The partial blocking activities of elicited antibodies against either the HAP2-GCS1 domain or the cd loop of this antigen have been recorded to hinder the transmission of Plasmodium species in Anopheles mid-gut. Thus, the objective of the present study was to investigate if the cd loop-fusion can enhance the quantity and quality of humoral and cellular immune responses against Plasmodium falciparum GCS1 in comparison to non-fusion antigen (without cd loop), in the adjuvanted and non-adjuvanted mouse groups. The immunogenicity of two constructs of P. falciparum generative cell specific 1 antigen, a fusion protein composed of cd loop and HAP2-GCS1 domain (cd-HAP) and another recombinant PfGCS1 containing solo HAP2-GCS1 domain (HAP2) were assessed to impede Plasmodium gametocytes integration before zygote formation. The antibodies profiling, titer, and avidity of induced antibodies were measured by the immunized mice sera, and the released cytokines (IL-5, TNF, and INF-γ) were analyzed in the supernatants of stimulated splenocytes. Furthermore, the inhibitory potency of the elicited antibodies against HAP2 and cd-HAP was measured during oocyst development by Standard Membrane Feeding Assay (SMFA). The comparative results in the present study showed the higher titer of IgG antibodies and IgG2a subclass, avidity, and transmission-reducing activity (TRA = 72.5 %) when mice were immunized by cd-HAP rather than HAP2. Moreover, our findings confirmed intensified Th1-directed immune responses in group 4 received cd-HAP/Poly(I:C). These findings declared the potential ability of cd loop fusion (cd-HAP) to upsurge humoral and cellular immune responses. However, the immune responses may switch to stronger Th1-type using alternative formulations. Explicitly, the cd-HAP-based vaccine may enhance the overall efficiency of immune responses and present a promising implementation in aiming malaria transmission., Competing Interests: Declaration of competing interest The article entitled “Cd loop fusion enhances the immunogenicity and the potential transmission blocking activity of Plasmodium falciparum Generative Cell Specific 1 (GCS1) antigen” has not been previously published elsewhere in any language and is not currently under consideration by any other publication. The authors also declare that they contributed significantly to the review, i.e. drafting, critical revision of the article, and also approval of the version to be published., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A novel bacterial display vector based on Escherichia coli has been engineered for recombinant protein production and purification. Accordingly, a construct harboring the enhanced green fluorescent protein (EGFP) and the ice nucleation protein (INP) was designed to produce EGFP via the surface display in E. coli cells. The fusion EGFP-expressed cells were then investigated using fluorescence measurement, SDS- and native-PAGE before and after TEV protease digestion. The displayed EGFP was obtained with a recovery of 57.7 % as a single band on SDS-PAGE. Next, the efficiency of the cell surface display for mutant EGFP (EGFP S202H/Q204H) was examined in sensing copper ions. Under optimal conditions, a satisfactorily linear range for copper ions concentrations up to 10 nM with a detection limit of 0.073 nM was obtained for cell-displayed mutant EGFP (mEGFP). In the presence of bacterial cell lysates and purified mEGFP, response to copper was linear in the 2-10 nM and 0.1-2 μM concentration range, respectively, with a 1.3 nM and 0.14 μM limit of detection. The sensitivity of bacterial cell lysates and surface-displayed mEGFP in the detection of copper ions is higher than the purified mEGFP., 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.)

Lepidopteran pests frequently cause significant damage to Sunflowers (Helianthus annuus). In this study, the insect resistant fusion gene Cry1Ab-Vip3Af2 was transformed into sunflower by Agrobacterium-mediated transformation. A transgenic event, named MCPN-7, was selected and characterized for its high resistance to both yellow peach moth (Dichocrocis punctiferalis) and cotton bollworm (Helicoverpa armigera), two polyphagous pests feeding on various plants including sunflower. The neonates of both species feeding on MCPN-7 resulted to 100 % mortality within 72 h in laboratory bioassays. No significant damage caused by the two insects was observed in field trials of MCPN-7. ELISA analysis revealed that the fusion protein was predominantly expressed in leaves, seeds and heads. The flanking genomic sequence of the T-DNA of the event MCPN-7 was determined and confirmed by PCR analysis. In conclusion, the transgenic sunflowers obtained in this study is highly resistant to wide spectrum of Lepidopteran insect pests and could potentially be a candidate event for commercial development., 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.)

Most of currently available sandwich-type enzyme-linked immunosorbent assays (ELISA) require the use of full-length animal-derived antibodies which poses welfare criticisms and are often expensive to produce. There is therefore a strong demand for the development of more affordable and animal-free methods to produce antibodies for sandwich ELISA assay. To address these issues, we propose here the development of a new technology based on two complementary rabbit single-chain variable fragments (scFvs) and an Ig-binding domain of protein L (PpL1) fused to a polystyrene-binding peptide (PS-tag) that can be recombinantly produced in bacteria. Toward this goal, we developed a rabbit scFv capable to bind the antigen via its variable regions while engaging protein L through its constant framework domain. To enhance the density of captured scFv and enable a better solvent exposure, we generated multiple PpL1 variants bearing polystyrene-binding peptides (PS) tags fused to its ends. The tandem trimer of PpL1 variant bearing PS-tags located at the N-terminus (PpL1'-T-PSN) revealed increased antigen-binding signal when immobilized on hydrophilic polystyrene (phi-PS) plates. By CDR-grafting different antigen-binding specificities into our engineered protein L-binding scFv we validated our technology against a different antigen. Finally, to further enhance the sensitivity of our assay, we implemented a protein L-based pretreatment to remove potential inhibitory immunoglobulin often present in the blood samples. The ability to rapidly and cost-effectively generate animal-free recombinant antibody fragments that can be adsorbed and specifically oriented on plates while retaining their antigen-binding properties could lead to the development of innovative and widely applicable sandwich ELISA systems for the efficient, versatile and sensitive detection of different types of antigens., (Copyright © 2024. Published by Elsevier B.V.)

Porphyra is one of the most economically valuable species of red algae, with porphyran being its primary bioactive polysaccharide. Highly active enzymes play a significant role in the research and development of porphyran. This study identified a PKD domain within a polysaccharide-binding protein, displaying an apparent molecular weight (M w ) of 20.20 kDa that is approximately twice the theoretical value, thereby suggesting the possibility of self-aggregation. By fusing it with porphyranase Por16B_Wf, a chimeric enzyme PKD-Por16B was constructed. It was confirmed that the fusion enzyme successfully assembles into an aggregation under the mediation of PKD domain, with its apparent M w (65.13 kDa) significantly higher than theoretical M w (46.02 kDa). The activity of PKD-Por16B was remarkably enhanced from 65.31 U/mg to 325.69 U/mg, accompanied by an improvement in enzymatic stability. Meanwhile, the hydrolysis pattern of PKD-Por16B remained unaltered in comparison to that of Por16B_Wf, indicating no significant deviation in its substrate specificity or reaction mechanism. These results suggest the feasibility of a strategy based on domain-induced aggregation to enhance enzyme activity, which is easy and economical., 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 B.V. All rights reserved.)

Based on the principle of cascade reaction, a fusion enzyme of dextransucrase and dextranase was designed without linker to catalyze the production of oligo-dextran with homogeneous molecular weight from sucrose in one catalytic step. Due to the different effects of temperature on the two components of the fusion enzyme, temperature served as the "toggle switch" for the catalytic efficiency of the two-level fusion enzyme, regulating the catalytic products of the fusion enzyme. Under optimal conditions, the fusion enzyme efficiently utilized 100 % of the sucrose, and the yield of oligo-dextran with a homogeneous molecular weight reached 70 %. The product has been purified and characterized. The probiotic potential of the product was evaluated by analyzing the growth of 10 probiotic species. Its cytotoxic and anti-inflammatory activities were also determined. The results showed that the long-chain oligo-dextran in this study had significantly better probiotic potential and anti-inflammatory activity compared to other oligosaccharides. This study provides a strategy for the application of oligo-dextran in the food and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A robust and stable carbonic anhydrase (CA) system is indispensable for effectively sequestering carbon dioxide to mitigate climate change. While microbial surface display technology has been employed to construct an economically promising cell-displayed CO 2 -capturing biocatalyst, the displayed CA enzymes were prone to inactivation due to their low stability in harsh conditions. Herein, drawing inspiration from biomineralized diatom frustules, we artificially introduced biosilica shell materials to the CA macromolecules displayed on Escherichia coli surfaces. Specifically, we displayed a fusion of CA and the diatom-derived silica-forming Sil3K peptide (CA-Sil3K) on the E. coli surface using the membrane anchor protein Lpp-OmpA linker. The displayed CA-Sil3K (dCA-Sil3K) fusion protein underwent a biosilicification reaction under mild conditions, resulting in nanoscale self-encapsulation of the displayed enzyme in biosilica. The biosilicified dCA-Sil3K (BS-dCA-Sil3K) exhibited improved thermal, pH, and protease stability and retained 63 % of its initial activity after ten reuses. Additionally, the BS-dCA-Sil3K biocatalyst significantly accelerated the CaCO 3 precipitation rate, reducing the time required for the onset of CaCO 3 formation by 92 % compared to an uncatalyzed reaction. Sedimentation of BS-dCA-Sil3K on a membrane filter demonstrated a reliable CO 2 hydration application with superior long-term stability under desiccation conditions. This study may open new avenues for the nanoscale-encapsulation of enzymes with biosilica, offering effective strategies to provide efficient, stable, and economic cell-displayed biocatalysts for practical applications., 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.)

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein., 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.)

Fasciolosis, caused by the liver fluke Fasciola gigantica, is a major parasitic disease that affects livestock and therefore causes significant economic losses in tropical countries. Although anthelminthic drugs can kill the parasite, drug-resistant liver fluke populations are increasing. In this study, a recombinant F. gigantica chimeric protein (rFgCHI) consisting of cathepsin L1H (FgCL1H), cathepsin B3 (FgCB3), and Saposin-like protein 1 (FgSAP1) was designed and expressed in Escherichia coli (BL21). The molecular weight of rFgCHI was 61 kDa. To study the antibody response, male BALB/c mice were immunized via the subcutaneous injection of rFgCHI combined with Quil A. Immunization with rFgCHI showed the induction of IgG1 and IgG2a with a higher IgG1 isotype level, indicating the potential of mixed Th1/Th2 immune responses, with Th2 predominating. However, the results showed high levels of IgG against the single proteins, except for rFgSAP1. Through Western blotting, mouse anti-rFgCHI polyclonal antibodies could be detected to the native proteins obtained from the parasite at all stages. Immunolocalization also revealed that the anti-rFgCHI antibodies could detect targeted antigens in the cecal epithelium of the parasite. These results demonstrated that rFgCHI is immunogenic to the mouse immune system and may potentially be a protein candidate for the development of a fasciolosis vaccine., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pornanan Kueakhai reports financial support was provided by Thailand Research Fund. Pornanan Kueakhai has patent #2303003287 pending to Thailand Science Research and Innovation. 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.)

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography-mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level., Competing Interests: Declaration of competing interest C. Buckley and C. MacHale are employees of Eli Lilly Kinsale Ltd. 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Enzyme-driven recycling of PET has now become a fully developed industrial process. With the right pre-treatment, PET can be completely depolymerized within workable timeframes. This has been realized due to extensive research conducted over the past decade, resulting in a large set of engineered PET hydrolases. Among various engineering strategies to enhance PET hydrolases, fusion with binding domains has been used to tune affinity and boost activity of the enzymes. While fusion enzymes have demonstrated higher activity in many cases, these results are primarily observed under conditions that would not be economically viable at scale. Furthermore, the wide variation in PET substrates, conditions, and combinations of PET hydrolases and binding domains complicates direct comparisons. Here, we present a self-consistent and thorough analysis of two leading PET hydrolases, LCC ICCG and PHL7. Both enzymes were evaluated both without and with a substrate-binding domain across a range of industrially relevant PET substrates. We demonstrate that the presence of a substrate-binding module does not significantly affect the affinity of LCC ICCG and PHL7 for PET. However, significant differences exist in how the fusion enzymes act on different PET substrates and solid substrate loading, ranging from a 3-fold increase in activity to a 6-fold decrease. These findings could inform the tailoring of enzyme choice to different industrial scenarios., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial or personal relationships that would influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrap TM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli. This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli. Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development., 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.)

Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni 2+ -NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni 2+ , Mg 2+ , and Na + inhibited the keratinolytic activity, Cu 2+ , Ca 2+ , and Mn 2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries., 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.)

Insect olfactory receptors (ORs) are seven-transmembrane domain ion channels that function by forming heteromeric complexes with olfactory receptor co-receptors (Orcos). In this study, we investigated the potential for enhancing sensitivity of odor detection and responsivity through genetic modification of Orcos, considering its wider application in odor sensing. First, we measured the intensity of response to 1-octen-3-ol for the mosquito Aedes aegypti OR (AaOR8) when complexed individually with an Orco from the same mosquito (AaOrco), the honeybee Apis mellifera (AmOrco), the silkworm Bombyx mori (BmOrco), or the fruit fly Drosophila melanogaster (DmOrco). Relative to the other Orcos, AmOrco demonstrated higher sensitivity and responsivity, with a 1.8 to 21-fold decrease in the half-maximal effective concentration (EC 50 ) and a 1.6-8.8-fold increase in the maximal effect (E max ), respectively. Furthermore, AmOrco co-expressed with AaOR10, BmOR56, or DmOR47a showed higher sensitivity and responsivity than AaOrco, BmOrco, or DmOrco co-expressed with their respective ORs. To further increase sensitivity and responsivity, we engineered chimeric Orcos by fusing AmOrco with DmOrco, considering the domain characteristics of Orcos. The response to 1-octen-3-ol was evaluated for AaOR8 when complexed individually with AmOrco, as well as for a mutant that combines DmOrco from the N-terminal (NT) to the C-terminal region of the fourth transmembrane domain (TM4) with the region of AmOrco following TM4 (Dm[NT-TM4]AmOrco). When compared to AmOrco, Dm(NT-TM4)AmOrco showed higher sensitivity and responsivity, with a 1.4-fold decrease in the EC 50 and a 1.4-fold increase in the E max , respectively. In addition, Dm(NT-TM4)AmOrco co-expressed with either DmOR47a or BmOR56 demonstrated higher sensitivity and responsivity than AmOrco co-expressed with their respective ORs. These results suggest that AmOrco could be a relatively more sensitive Orco, and further enhancement of sensitivity and responsivity could be achieved through recombination with heterologous Orcos near the TM4 of AmOrco., 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 emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses., 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.)

Abstract: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase., 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 elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human interferon α2 (IFNα2) is a cytokine with broad-spectrum antiviral activity, and its engineered forms are widely used to treat viral infections. However, IFNα2 may trigger proinflammatory responses and underlying side effects during treatment. Trefoil factor 2 (TFF2) is a secreted protein with anti-inflammatory properties. Here, we explored whether coupling IFNα2 to TFF2 in a two-in-one fusion form could combine the beneficial effects of both molecules on viral infections toward a more desirable treatment outcome. We engineered two forms of human IFNα2 and TFF2 fusion proteins, IFNα2-TFF2-Fc (ITF) and TFF2-IFNα2-Fc (TIF), and examined their properties in vitro in comparison to IFNα2 and TFF2 alone. RNA-Seq was further used to explore such comparison on dynamic gene regulation at transriptomic level. These in vitro assessments collectively indicated that TIF largely retained the antiviral activity of IFNα2 while being a weaker inflammation inducer, consistent with the presence of TFF2 activity. We further demonstrated the superiority of TIF over IFNα2 or TFF2 alone in treating influenza infection using a mouse infection model. Together, our study provided evidence supporting that, by possessing antiviral activity conferred by IFNα2 with complementation from TFF2 in suppressing the inflammatory side effects, the fusion proteins, particularly TIF, represent more effective agents against influenza and other respiratory viral infections than IFNα2 or TFF2 alone. It implies that merging two molecules with complementary functions holds potential for developing novel therapeutics against viral infections., 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.)

To enhance the DNA/RNA amplification efficiency and inhibitor tolerance of Bst DNA polymerase, four chimeric Bst DNA polymerase by fusing with a DNA-binding protein Sto7d and/or a highly hydrophobic protein Hp47 to Bst DNA polymerase large fragment. One of chimeric protein HpStBL exhibited highest inhibitor tolerance, which retained high active under 0.1 U/μL sodium heparin, 0.8 ng/μL humic acid, 2.5× SYBR Green I, 8 % (v/v) whole blood, 20 % (v/v) tissue, and 2.5 % (v/v) stool. Meanwhile, HpStBL showed highest sensitivity (93.75 %) to crude whole blood infected with the African swine fever virus. Moreover, HpStBL showed excellent reverse transcriptase activity in reverse transcription loop-mediated isothermal amplification, which could successfully detect 0.5 pg/μL severe acute respiratory syndrome coronavirus 2 RNA in the presence of 1 % (v/v) stools. The fusion of two domains with different functions to Bst DNA polymerase would be an effective strategy to improve Bst DNA polymerase performance in direct loop-mediated isothermal amplification and reverse transcription loop-mediated isothermal amplification detection, and HpStBL would be a promising DNA polymerase for direct African swine fever virus/severe acute respiratory syndrome coronavirus 2 detection due to simultaneously increased inhibitor tolerance and reverse transcriptase activity., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interest associated with this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Fc-fused receptor binding domain (RBD-Fc) vaccine for SARS-CoV-2 has garnered significant attention for its capacity to provide effective and specific immune protection. However, its immunogenicity is limited, highlighting the need for improvement in clinical application. Nanoparticle delivery has been shown to be an effective method for enhancing antigen immunogenicity. In this study, we developed bivalent nanoparticle recombinant protein vaccines by assembling the RBD-Fc of SARS-CoV-2 and Fc-binding homo-oligomers o42.1 and i52.3 into octahedral and icosahedral nanoparticles. The formation of RBD-Fc nanoparticles was confirmed through structural characterization and cell binding experiments. Compared to RBD-Fc dimers, the nanoparticle vaccines induced more potent neutralizing antibodies (nAb) and stronger cellular immune responses. Therefore, using bivalent nanoparticle vaccines based on RBD-Fc presents a promising vaccination strategy against SARS-CoV-2 and offers a universal approach for enhancing the immunogenicity of Fc fusion protein vaccines., 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.)

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein., 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.)

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a C C -intein tag is engineered into a protein of interest for binding to a N C -intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the N C -intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the C C -intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Johan Ohman has patent #WO2021099607A1 licensed to CYTIVA BIOPROCESS R&D AB. 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. Published by Elsevier B.V.)

Chimeric antigen receptor T (CAR-T) cell therapy is regarded as a potent immunotherapy and has made significant success in hematologic malignancies by eliciting antigen-specific immune responses. However, response rates of CAR-T cell therapy against solid tumors with immunosuppressive microenvironments remain limited. Co-engineering strategies are advancing methods to overcome immunosuppressive barriers and enhance antitumor responses. Here, we engineered an IL-2 mutein co-engineered CAR-T for the improvement of CAR-T cells against solid tumors and the efficient inhibition of solid tumors. We equipped the CAR-T cells with co-expressing both tumor antigen-targeted CAR and a mutated human interleukin-2 (IL-2m), conferring enhanced CAR-T cells fitness in vitro, reshaped immune-excluded TME, enhanced CAR-T infiltration in solid tumors, and improved tumor control without significant systemic toxicity. Overall, this subject demonstrates the universal CAR-T cells armed strategy for the development and optimization of CAR-T cells against solid tumors., 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. Published by Elsevier B.V.)

Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nef mut -Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nef mut -Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nef mut -Tat combined with exosomes harboring Hsp70 (Exo-Nef mut -Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nef mut -Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure., 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.)

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications., 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. Published by Elsevier B.V.)

Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors., 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 © 2023 Elsevier Ltd. All rights reserved.)

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli , achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC 50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane β-barrels, duplication is visible with β-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane β-barrels. Specifically, the topology of some 16- and 18-stranded β-barrels appear to have evolved through a loop to β-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded β-barrel and an evolutionarily related 16-stranded β-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane β-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to β-hairpin transition., 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In conventional immunoassays, a secondary antibody is used to amplify the signal generated by the binding of the primary antibody to the target analyte. Due to concerns regarding animal use and cost-inefficiency of secondary antibody productions, there is a significant demand for the development of recombinant secondary antibody mimics (rSAMs). Here, we developed rSAMs using a signal-generating enzyme, monomeric alkaline phosphatase (mALP), and antibody-binders, including monomeric streptavidin (mSA2) and mouse IgG1- or rabbit IgG-binding nanobodies (MG1Nb or RNb). The mALP-MG1Nb, mALP-RNb, and mALP-mSA2 were genetically constructed and produced in large quantities using bacterial overexpression systems, which reduced manufacturing costs and time without the use of animals. Each rSAM exhibited high and selective binding to its respective primary antibody, generating linear band signals corresponding to the amounts of target analytes in western blots. The rSAMs also successfully generated sigmoidal signal curves that increased as the sample concentration increased. Moreover, they generated stronger signals than conventional ALP-conjugated secondary antibodies and SA, particularly in the medium to high sample concentration range, in both indirect and sandwich-type indirect ELISAs at the same sample concentration. The rSAMs we developed here may provide new insights to develop novel immunoassay-based analytical and diagnostic tools., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sebyung Kang reports financial support was provided by National Research Foundation of Korea. Sebyung Kang reports financial support was provided by Ulsan National Institute of Science and Technology., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Serological tests developed for COVID-19 diagnostic are based on antibodies specific for SARS-CoV-2 antigens. Most of the antigens consist of a fragment or a whole amino acid sequence of the nucleocapsid or spike proteins. We evaluated a chimeric recombinant protein as an antigen in an ELISA test, using the most conserved and hydrophilic portions of the S1-subunit of the S and Nucleocapsid (N) proteins. These proteins, individually, indicated a suitable sensitivity of 93.6 and 100% and a specificity of 94.5 and 91.3%, respectively. However, our study with the chimera containing S1 and N proteins of SARS-CoV-2 suggested that the recombinant protein could better balance both the sensitivity (95.7%) and the specificity (95.5%) of the serological assay when comparing with the ELISA test using the antigens N and S1, individually. Accordingly, the chimera showed a high area under the ROC curve of 0.98 (CI 95% 0.958-1). Thus, our chimeric approach could be used to assess the natural exposure against SARS-CoV-2 virus over time, however, other tests will be necessary to better understand the behaviour of the chimera in samples from people with different vaccination doses and/or infected with different variants of the virus., (Copyright © 2023. Published by Elsevier B.V.)

Alka(e)nes are high-value chemicals with a potentially broad range of industrial applications because of their following advantages: (1) chemical and structural resemblance to petroleum hydrocarbons and (2) higher energy density and hydrophobicity than those of other biofuels. The low yield of bio-alka(e)nes, however, hinders their commercial application. The activity and solubility of acyl carrier protein (ACP) reductase (AAR) affect alka(e)ne biosynthesis in cyanobacteria. The enhancement of the activity and concentration of soluble AAR through genetic and process engineering can improve bio-alka(e)ne yield. Although fusion tags are used to enhance the expression or solubility of recombinant proteins, their effectiveness in improving the production of bio-alka(e)nes has not yet been reported. Fusion tags can be used to improve the amount or activity of soluble AAR in Escherichia coli and to increase the yield of alka(e)nes in E. coli cells co-expressing aldehyde deformylating oxygenase (ADO). Hence, in the present study, histidine (His 6 /His 12 ), thioredoxin (Trx), maltose-binding protein (MBP), and N-utilization substance (NusA) were used as AAR fusion tags. The strain expressing SeAAR with His 12 tag and NpADO showed a 7.2-fold higher yield of alka(e)nes than the strain expressing AAR without fusion tag and NpADO. The highest titer of alka(e)nes (194.78 mg/L) was achieved with the His 12 tag., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

CCL21, a secondary lymphoid tissue chemokine, plays an important role in generating an effective anti-tumor immune response. In this study, a genetically modified CCL21 was developed by inserting a pH low insertion peptide to establish a CCL21-rich microenvironment for tumors. The fusion tag thioredoxin (Trx) was designed and fused at the N-terminal of the recombinant protein to protect it from being irrevocably misfolded in microbial host cells. The prokaryotic expression vector pET32a-CCL21-pHLIP was constructed and successfully expressed in E. coli BL21 (DE3) with a soluble expression form and a molecular weight of ~35 kDa. The induction conditions were optimized to obtain an extremely high yield of 6.7 mg target protein from 31.1 mg total protein. The 6xHis tagged Trx-CCL21-pHLIP was purified using Ni-NTA resin, and it was confirmed using SDS-PAGE and Western blot analyses. Consequently, the Trx-CCL21-pHLIP protein was successfully displayed on the cancer cell surface in a weak acidic microenvironment and showed the same ability as CCL21 in recruiting CCR7-positive cells. Additionally, the CCL21 fusion protein with or without Trx tag showed similar functions. Therefore, the study implies the feasibility of directing a modular genetic method for the development of protein-based drugs., 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 © 2023 Elsevier B.V. All rights reserved.)