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Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies., Competing Interests: Declaration of Competing Interest JC and JB were founders of AlbaJuna Therapeutics. AB, JB, JC and FC were employees of AlbaJuna therapeutics. Other authors, no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. 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.)

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CD59 is a cell-surface inhibitor of the terminal step in the complement cascade. However, in addition to its complement inhibitory function, a non-canonical role of CD59 in pancreatic beta cells has been identified. Two recently discovered intracellular alternative splice forms of CD59, IRIS-1 and IRIS-2, are involved in insulin exocytosis through interactions with SNARE-complex components. In mice, the CD59 gene has undergone duplication and to further explore the role of CD59 in insulin secretion, blood glucose homeostasis was studied in a CD59 double knockout (CD59abKO) mouse model. However, no phenotypic deviation related to insulin secretion or blood glucose homeostasis was observed for the CD59abKO mice. Instead, a CD59ba hybrid transcript formed as a consequence of the mutation induced to generate the model was identified. This hybrid transcript is expressed in pancreatic islets of the CD59abKO mice and is comprised of the remaining exons of the two CD59 genes spliced together. Similar to canonical CD59, the CD59ba hybrid was found to be glycosylated and present on the cell surface when exogenously expressed in INS-1 832/13 cells. Furthermore, INS-1 832/13 cells over-expressing the mouse CD59ba hybrid retained normal insulin secretion following siRNA-mediated knockdown of canonical CD59. Hence, although the CD59ba hybrid has lost the complement inhibitory function, the intracellular insulin secretory function remains. These results provide further information concerning the structural requirements of CD59 in its intracellular role relative to its role as a complement inhibitor. It also highlights the importance of carefully assessing plausible consequences of induced mutations in research models., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Since 1982, recombinant insulin has been used as a substitute for pancreatic insulin from animals. However, increasing demand in medical and food industries warrants the development of more efficient production methods. In this study, we aimed to develop a novel and efficient method for insulin production using a yeast secretion system., Methods: Here, insulin C-peptide was replaced with a hydrophilic fusion partner (HL18) containing an affinity tag for the hypersecretion and easy purification of proinsulin. The HL18 fusion partner was then removed by in vitro processing with the Kex2 endoprotease (Kex2p), and authentic insulin was recovered via affinity chromatography. To improve the insulin functions, molecular chaperones of the host strain were reinforced via the constitutive expression of HAC1., Results: The developed method was successfully applied for the expression of cow, pig, and chicken insulins in yeast. Moreover, biological activity of recombinant insulins was confirmed by growth stimulation of cell line., Conclusions: Therefore, replacement of the C-peptide of insulin with the HL18 fusion partner and use of Kex2p for in vitro processing of proinsulin guarantees the economic production of animal insulins in yeast., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that select magnetosome proteins may associate in mammalian cells., (© 2024. The Author(s).)

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.)

N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (K m  = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management., 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 Ltd.)

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for selection via multiple display methods. This protocol describes selection from affibody libraries by Escherichia coli cell surface display. With this method, high-diversity libraries of 10 11 can be displayed on the cell surface. The method involves two steps for selection of binders from high-diversity libraries: magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is used first to enrich the library in target-binding clones and to decrease diversity to a size that can be effectively screened and sorted in the flow cytometer in a reasonable time (typically <10 7 cells). The protocol is based on methodology using an AIDA-I autotransporter for display on the outer membrane, but the general procedures can also be adjusted and used for other types of autotransporters or alternative E. coli display methods., (© 2024 Cold Spring Harbor Laboratory Press.)

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.)

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library. This is then followed by amplification of the library, which can then be used for biopanning using multiple methods. This protocol describes amplification of affibody libraries, followed by biopanning using phage display and analysis of the selection output. The general procedure is mainly for selection of first-generation affibody molecules from large naive (unbiased) libraries, typically yielding affibody hits with affinities in the low nanomolar range. For selection from affinity maturation libraries with the aim of isolating variants of even higher affinities, the procedure is similar, but parameters such as target concentration and washing are adjusted to achieve the proper stringency., (© 2024 Cold Spring Harbor Laboratory Press.)

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed., (© 2024 Cold Spring Harbor Laboratory Press.)

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization., (© 2024 The Author(s).)

Algae has been proven to have the potential to be efficient biosorbents in the detection and remediation of heavy metal pollution such as cadmium in the environment. This study aims to enhance the cadmium adsorption capacity of Chlamydomonas reinhardtii by expressing the cadmium-binding protein CADR on the cell wall by the surface display technology. Firstly, the golden gate technique was employed to construct the transformation vector PET-X- CADR , which anchored CADR to the cell wall with the cell wall protein GP1. The high-throughput screening with the fluorescence signal of the fusion tag YFP resulted in three engineered algal strains with high expression of CADR on the cell wall. Physiological experiments demonstrated that the CADR displayed on the cell wall did not affect the growth of the engineered algal strains exposed to cadmium with the concentration below 200 μmol/L. In the presence of 200 μmol/L cadmium, the growth rates of CADR-engineered algal strains were three times as that of the wild-type, indicating stronger tolerance of the CADR-engineered algae to cadmium. The protein lyase GLE released during the mating of Chlamydomonas was used to isolate the cell walls of wild-type and engineered strains, the cadmium content of which was compared. The results showed that the cell wall of the engineered strain exhibited an increase of 33% in cadmium adsorption capacity. This study gives insights into the application of algae in the management of cadmium pollution in the environment, especially in the recycling of heavy metals from the environment.

Polyethylene terephthalate (PET) is a largely-produced polymer worldwide. However, its extensive waste generation and resistance to degradation pose significant environmental concerns. Consequently, there is considerable interest in researching enzymatic degradation of PET. Relevant studies have shown that the addition of a carbohydrate binding module (CBM) can increase the affinity between the enzyme and the substrate, enhancing the enzyme's degradation ability. In order to develop more efficient PET hydrolytic enzymes, this study introduced carbohydrate binding domains (CBMs) from different families with different substrate affinities into the PET-degrading enzyme LCC-ICCG. High crystallinity PET powder and amorphous PET film were used as substrates to characterize the degradation efficiency of the modified enzymes, aiming to explore the enzyme with the optimal degradation ability. The results showed that the fusion of type B CBM reduced the degradation rate and T m value of the enzyme towards PET, while the introduction of type A and type C CBMs significantly improved the degradation rate of the enzyme towards the film-like substrate. The degradation rate and T m value of PET were also enhanced, especially with the fusion enzyme LCC-ICCG-CBM9-2, which showed an over 10-fold increase in the degradation rate compared to the original enzyme LCC-ICCG. Therefore, this study demonstrates that by introducing type A and type C CBMs, the degradation rate and thermal stability of LCC-ICCG towards film-like PET can be improved, addressing the issue of its low activity and enabling more effective PET degradation. This research provides support for plastic degradation technology and contributes to environmental conservation efforts.

Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion-powered motors with more distant homology. Here, we synthesized chimeras of MotAB, PomAPotB, and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimized by directed evolution. Whole-genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with the rational design of chimeric IRMs., Importance: Ion-powered rotary motors (IRMs) underpin the rotation of one of nature's oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors., Competing Interests: The authors declare no conflict of interest.

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1-97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H6-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H6-trC1INH(MGS)-MSA), and H6-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H6-trC1INH(MGS)-MSA was significantly increased versus that of H6-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H6-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H6-trC1INH(MGS)-MSA by 33% relative to that of H6-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H6-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sivananthan 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.)

Non-immunoglobulin-based scaffold proteins (SPs) represent one of the key therapeutic target-specific and high-affinity binders in modern medicine. Among their cellular targets are signaling receptors, in particular, receptor tyrosine kinases, whose dysfunction leads to the development of cancer and other serious diseases. Successful applications of SPs have been reported for HER receptor type 2 (HER2), a member of the human epidermal growth factor receptor family that regulates cell growth and differentiation. To extend the blood residence of SPs and prevent their high accumulation in the kidneys, these proteins are often fused with serum albumin. Promising results for HER2-binding activity were obtained for SP G3 from the DARPins (Designed Ankyrin Repeat Proteins) family fused with an albumin-binding domain (ABD). Interestingly, the detected HER2-G3 binding strongly depended on the position of the G3 module in the sequence of the constructs. Further improvement of these constructs for biomedical applications requires deciphering the molecular mechanism responsible for this effect. Here, we investigate the structural and dynamic aspects of ABD-G3 and G3-ABD chimeras using NMR spectroscopy and molecular modeling. Based on biophysical data, we come to the conclusion that extensive inter-domain contacts form in both constructs, although their binding interfaces and complex stability are somewhat different. Also, it is shown that the domain linker plays an important role-it limits the accessibility of the detected protein-protein binding sites, depending on the order of the domains in the chimeric molecules. These results create a solid structural basis for the rational design of new effective SP constructs targeting the signaling receptors in cells.

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans-Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) plays an essential role in glycolysis and in the antioxidant pathway associated with glutathione. Therefore, we investigated the effects of PFKFB3 on oxidative and ischemic damage. We synthesized a fusion protein of transactivator of transcription (Tat)-PFKFB3 to facilitate its passage into the intracellular space and examine its effects against oxidative stress induced by hydrogen peroxide (H 2 O 2 ) treatment and ischemic damage caused by occlusion of the common carotid arteries for 5 min in gerbils. The Tat-PFKFB3 protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, with higher levels observed 18 h after treatment. Furthermore, treatment with 6 µM Tat-PFKFB3 demonstrated intracellular delivery into HT22 cells, as analyzed through immunocytochemical staining. Moreover, it significantly ameliorated the reduction of cell viability induced by 200 µM H 2 O 2 treatment. Tat-PFKFB3 treatment also alleviated H 2 O 2 -induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, the intraperitoneal administration of 2 mg/kg Tat-PFKFB3 efficiently delivered the substance to all hippocampal areas, including the hippocampal CA1 region. This administration significantly mitigated ischemia-induced hyperlocomotion, long-term memory deficits, and ischemic neuronal death in the hippocampal CA1 region after ischemia. Additionally, treatment with 2 mg/kg Tat-PFKFB3 significantly ameliorated the translocation of Cdk5 from the cytosol to the nucleus in the hippocampal CA1 region 24 h after ischemia, but not in other regions. The treatment also significantly reduced reactive oxygen species formation in the CA1 region. These findings suggest that Tat-PFKFB3 reduces neuronal damage in the hippocampal CA1 region after ischemia through the reduction of Cdk5 signaling and reactive oxygen species formation. Therefore, Tat-PFKFB3 may have potential applications in reducing ischemic damage., (© 2024. The Author(s).)

Extracellular vesicles (EVs) are attracting growing attention for therapeutic use and as diagnostic markers, particularly for cancer. Although therapies based on small interfering RNAs are under intensive research, other therapeutic molecules, especially proteins, have not been sufficiently investigated. One of the major method for loading proteins into EVs is electroporation; however, it damages membrane integrity and requires repeated purification, precluding clinical applications. Thus, natural and efficient protein transfer is a prerequisite for the clinical application of protein-based EV therapy. Another prerequisite is an efficient endosomal escape, as most EVs incorporated into receptor cells result in endosomal degradation. Therefore, we generated a short CD9 (sCD9)-INF/TAT tag for efficiently transfers fused proteins to the EV and enhances endosomal escape to address the abovementioned problems. Interestingly, protein transfer via EVs drastically improved when the EV producer and receptor cells were cocultured, strongly indicating bystander effects of cells producing therapeutic proteins fused with a sCD9-INF/TAT tag. This method can be applied to a wide range of therapeutic technologies, including cellular transplantation or viral therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Inano, Kitano. 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.)

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Multifunctional enhanced collagen materials in green biomanufacturing are highly desired yet challenging due to the poor comprehensive performance caused by the adoption of targeting monofunctional peptides. Herein, novel collagen analog design strategy using multicopy tandem of mucin-type sequence (GAPGAPGSQGAPGLQ) derived from human COL1α1 to construct basic building blocks is reported, in which SUMO tag is added to the N-terminal of the protein as a stabilizing core. In particular, novel collagen analogs (named S1506, S1511, S1523, and S1552) with multicopy mucin-type sequences (repeated 6, 11, 23, and 52 times), which were constructed in Escherichia coli , have distinct orientation preferences of functional enhancement (including cell proliferation, differentiation, migration, antioxidant activity, and anti-inflammatory property) compared to COL1α1 in HaCaT and THP-1 cell experiments due to variant three-dimensional structures (the different-length mucin-type polypeptide chains wind around central SUMO tag). Our findings suggest that the innovative protein design and synthesis approaches employed in the construction of these novel S15 proteins have the potential to advance the development of new types of recombinant collagen analogs.

Hippocampal neurons can be the first to be impaired with neurodegenerative disorders, including Alzheimer's disease (AD). Most drug candidates for causal therapy of AD cannot either enter the brain or accumulate around hippocampal neurons. Here, we genetically engineered insulin-fusion proteins, called hippocampal neuron-targeting (Ht) proteins, for targeting protein drugs to hippocampal neurons because insulin tends to accumulate in the neuronal cell layers of the hippocampus. In vitro examinations clarified that insulin and Ht proteins were internalized into the cultured hippocampal neurons through insulin receptor-mediated macropinocytosis. Cysteines were key determinants of the delivery of Ht proteins to hippocampal neurons, and insulin B chain mutant was most potent in delivering cargo proteins. In vivo accumulation of Ht proteins to hippocampal neuronal layers occurred after intracerebroventricular administration. Thus, hippocampal neuron-targeting technology can provide great help for developing protein drugs against neurodegenerative disorders., Competing Interests: Competing interests statement:The authors declare no competing interest.

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting our ability to study CHO cell biology and detect host cell protein (HCP) impurities in the final antibody drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of eight commercial antibody drug products (7 mAbs and 1 Fc-fusion protein) using the extended protein sequence database revealed the presence of microprotein impurities. We present evidence that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation and the regulation of protein synthesis in CHO cell lines., (© 2024. The Author(s).)

Interferons (IFNs) are universally acknowledged for their pivotal role in antiviral and anticancer responses. Thus, the primary aim of our study was to explore the expressions of IFN-α1b, α2b, and gamma in tobacco leaves via agrobacterium-mediated transient transformation and investigate their possible activities. Briefly, fusion with green fluorescent protein tags aided in detecting the expressed IFN proteins in the foliar tissues. The genetic constructs encoding these fusion proteins were inserted into the MagnICON plant transient expression vector, followed by transformation into the Agrobacterium strain GV3101. The transformed bacteria were then used to infiltrate tobacco leaves. After post-infiltration, protein expression was confirmed within 72 h via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the fusion proteins were subsequently purified using high-performance liquid chromatography for identification. Both the antiviral and anticancer potencies of these IFN fusion proteins were evaluated using the WISH/VSV (WISH cells/Vesicular stomatitis virus) microneutralization and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. Results indicated robust expression of the targeted IFN genes in plant tissues and significant biological activities against pathogens and cancer cells. Consequently, this study substantiated the viability of producing these therapeutic proteins in plants, potentially revolutionizing the manufacture of interferons biologically.

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.)

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer., (© 2024 Wiley Periodicals LLC.)

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.)

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis., (© 2024 Merck Sharp & Dohme LLC and The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/μg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant., (Copyright © 2024 Elsevier Inc. 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.)

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.)

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH 4 ) 2 SO 4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. 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.)

Fibroblast growth factor 21 (FGF21) is pivotal in regulating energy metabolism, highlighting substantial therapeutic potential for non-alcoholic steatohepatitis (NASH). Previously, we reported a long-acting FGF21 fusion protein, PsTag-FGF21, which was prepared by genetically fusing human FGF21 with a 648-residue polypeptide (PsTag). While this fusion protein demonstrated therapeutic efficacy against NASH, our final product analysis revealed the presence of fixed impurities resistant to effective removal, indicating potential degradation of PsTag-FGF21. Here, we enriched and analyzed the impurities, confirming our hypothesis regarding the C-terminal degradation of PsTag-FGF21. We now describe a new variant developed to eliminate the C-terminal degradation. By introducing one mutation located at the C-terminal of PsTag-FGF21(V169L), we demonstrated that the new molecule, PsTag-FGF21(V169L), exhibits many improved attributes. Compared with PsTag-FGF21, PsTag-FGF21(V169L) displayed elevated bioactivity and stability, along with a twofold enhanced binding affinity to the coreceptor β-Klotho. In vivo, the circulating half-life of PsTag-FGF21(V169L) was further enhanced compared with that of PsTag-FGF21. In NASH mice, PsTag-FGF21(V169L) demonstrated efficacy with sustained improvements in multiple metabolic parameters. Besides, PsTag-FGF21(V169L) demonstrated the ability to alleviate NASH by decreasing hepatocyte apoptosis. The superior biophysical, pharmacokinetic, and pharmacodynamic properties, along with the positive metabolic effects, imply that further clinical development of PsTag-FGF21(V169L) as a metabolic therapy for NASH patients may be warranted., 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. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: W. Todd Miller reports financial support was provided by Veterans Health Administration. 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., (Published by Elsevier Inc.)

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para -azido- L -phenylalanine (pAzF) in Escherichia coli . First, we expressed the maltose-binding protein (MBP)-fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Microtubule (MT) consists of α-tubulin and β-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo , we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice ( Oryza sativa ) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7 , OsKCBP , OsCLASP , and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice., Competing Interests: The authors declare that they have no competing interests., (© 2024 Xu et al.)

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus . These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7 GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7 GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.

Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a 'biological Proteolysis-Targeting Chimera' (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling., (© 2024. The Author(s).)

This review discusses the current research progress in molecular farming technology in the field of skincare, with an emphasis on molecular farming expression strategies. The strategies of transdermal drug delivery and their advantages are also highlighted. The expression of cosmetically relevant fused proteins has become an important way to enhance the efficacy of the proteins. Therefore, we also discuss the feasibility and strategies for expressing fusion proteins in A. thaliana , specifically the fusion of Epidermal growth factor (EGF) to a cell-penetrating peptide (CPP), in which the production can be greatly enhanced via plant expression systems since these systems offer higher biosecurity, flexibility, and expansibility than prokaryotic, animal and mammalian expression systems. While the fusion of EGF to CCP can enhance its transdermal ability, the effects of the fusion protein on skin repair, melasma, whitening, and anti-aging are poorly explored. Beyond this, fusing proteins with transdermal peptides presents multiple possibilities for the development of tissue repair and regeneration therapeutics, as well as cosmetics and beauty products. As certain plant extracts are known to contain proteins beneficial for skin health, the expression of these proteins in plant systems will better maintain their integrity and biological activities, thereby facilitating the development of more effective skincare products., Competing Interests: The authors declare that they have no competing interests., (© 2024 Yu et al.)

Plastics are a cornerstone of the modern world, yet the durable material properties that we have come to depend upon have made them recalcitrant environmental pollutants. Biological solutions in the form of engineered enzymes offer low energy and sustainable approaches to recycle and upcycle plastic waste, uncoupling their production and end of life from fossil fuels and greenhouse gases. These enzymes however, encounter immense challenges acting on plastics: facing hydrophobic surfaces, molecular crowding, and high levels of substrate heterogeneity. There have been mixed reports about the benefits of fusing partner domains to polyethylene terephthalate (PET) degrading enzymes, with moderate improvements identified under specific conditions, but no clarity into the factors that underlie the mechanisms. Here, we use the SpyCatcher003:SpyTag003 technology, which demonstrates a profound 47 °C shift in T m upon irreversible complex formation, to investigate the influence of the thermal stability of the fusion partner on a range of PETases selected for their optimal reaction temperatures. We find that the thermal stability of the fusion partner does not have a positive correlation on the activity of the enzymes or their evident kinetic and thermal stabilities. Instead, it appears that the fusion to less stable SpyCatcher003 tends to increase the measured activation energy of unfolding compared to the more stable complex and wildtype enzymes. Despite this, the fusions to SpyCatcher003 do not show significantly better catalytic activity on PET films, with or without SpyTag003, and were found to be sometimes disruptive. The approach we highlight here, in using a fusion partner with controllable melting temperature, allowed us to dissect the impact of the stability of a fusion partner on enzyme properties. Although fusion stability did not appear to be coupled with identifiable trends in enzymatic activities, careful analysis of the unfolding pathways, and solid and solution activities of a wider range of enzymes may yield a more detailed understanding.

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 V L ) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli . The HaCad-TBR-2D3 V L enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 V L over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

Importin β-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUS IBB ), carrying the importin β binding (IBB) domain, is solubilized by importin β in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUS IBB replacing the aggregation-prone endogenous FUS., (© 2024. The Author(s).)

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture., 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.)