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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.)

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.)

The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.

Photodynamic therapy (PDT) is a noninvasive approach to cancer treatment, wherein cell death is initiated by singlet oxygen ( 1 O 2 ) production via energy transfer from excited photosensitizers to ground-state O 2 . Effective clinical photosensitizers necessitate water solubility for in vivo administration. Hydrophobic dyes, such as phthalocyanines, cannot be used directly as photosensitizers. Herein, we synthesized a myoglobin-(human serum albumin) fusion protein reconstituted with zinc-phthalocyanine (ZnPc), termed ZnPcMb-HSA. The photophysical properties of ZnPcMb-HSA closely resemble those of ZnPc-substituted Mb. Notably, ZnPc dissociates from ZnPcMb-HSA and selectively accumulates within cancer cells, while the protein components remain extracellular. Treatment of four distinct cell lines with ZnPcMb-HSA, followed by red-light irradiation, effectively induced apoptosis. The half-maximal inhibitory concentrations (IC 50 ) against these cancer cell lines ranged between 0.1-0.5 μM. Reconstituted Mb-HSA emerges as a promising carrier for transporting various water-insoluble porphyrinoid photosensitizer to target cancer cells in PDT applications., (© 2024 Wiley-VCH GmbH.)

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4 + cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand., 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.)

Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag-TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag-TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1 H NMR and 13 C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems., (© 2024 John Wiley & Sons Ltd.)

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus., 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.)

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest. This article does not contain any studies involving humans or animals as experimental objects., (Copyright © 2024. Published by Elsevier Inc.)

Influenza poses a substantial health risk, with infants and the elderly being particularly susceptible to its grave impacts. The primary challenge lies in its rapid genetic evolution, leading to the emergence of new Influenza A strains annually. These changes involve punctual mutations predominantly affecting the two main glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA). Our existing vaccines target these proteins, providing short-term protection, but fall short when unexpected pandemics strike. Delving deeper into Influenza's genetic makeup, we spotlight the nucleoprotein (NP) - a key player in the transcription, replication, and packaging of RNA. An intriguing characteristic of the NP is that it is highly conserved across all Influenza A variants, potentially paving the way for a more versatile and broadly protective vaccine. We designed and synthesized a novel NP-Hoc fusion protein combining Influenza A nucleoprotein and T4 phage Hoc, cloned using Gibson assembly in E. coli, and purified via ion affinity chromatography. Simultaneously, we explore the T4 coat protein Hoc, typically regarded as inconsequential in controlled viral replication. Yet, it possesses a unique ability: it can link with another protein, showcasing it on the T4 phage coat. Fusing these concepts, our study designs, expresses, and purifies a novel fusion protein named NP-Hoc. We propose this protein as the basis for a new generation of vaccines, engineered to guard broadly against Influenza A. The excitement lies not just in the immediate application, but the promise this holds for future pandemic resilience, with NP-Hoc marking a significant leap in adaptive, broad-spectrum influenza prevention., Competing Interests: Declaration of competing interest The authors declare no commercial or financial conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The widespread adoption of fast fashion has led to a significant waste problem associated with discarded textiles. Using proteins to color textiles can serve as a sustainable alternative to chemical dyes as well as reduce the demand for new raw materials. Here, we explore the use of chromogenic fusion proteins, consisting of a chromoprotein and a carbohydrate-binding module (CBM), as coloring agents for cellulose-based textiles such as cotton. We examined the color properties of chromoproteins AeBlue, SpisPink and Ultramarine alone and fused to CBM under various conditions. AeBlue, SpisPink and Ultramarine exhibited visible color between pH 4-9 and temperatures ranging from 4 to 45℃. Fusing CBM Clos from Clostridium thermocellum and CBM Ch2 from Trichoderma reesei to the chromoproteins had no effect on the chromoprotein color properties. Furthermore, binding assays showed that chromoprotein fusions did not affect binding of CBMs to cellulosic materials. Cotton samples bound with Ultramarine-Clos exhibited visible purple color that faded progressively over time as the samples dried. Applying 10% 8000 polyethylene glycol to cotton samples markedly preserved the color over extended periods. Overall, this work highlights the potential of chromoprotein-CBM fusions for textile dying which could be applied as a color maintenance technology or for reversible coloring of textiles for events or work wear, contributing to sustainable practices and introducing new creative opportunities for the industry., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01-pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01-pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2., Competing Interests: The authors declare that they have no competing interests., (© 2024 Yang et al.)

Association-controllable hemoprotein assemblies were constructed from a fusion protein containing two c-type cytochrome units using 3D domain swapping. The hemoprotein assembly exhibited a dynamic exchange between cyclic and linear structures and could be regulated by carbon monoxide (CO) and imidazole binding.

Background: Immobilized proteins hold promise as the basic units that have enabled a broad range of analytical applications within chemical measurement science. As yet, the co-immobilization of diverse proteins at precise ratio and whether they give rise to improved analytical performance remain challengeable. Herein, we utilized a circularly permuted HaloTag (cpHaloTag) to achieve the co-immobilization of two proteins at precise ratio, which was applied in developing a chromatographic method with improved specificity for pursuing dual-target compounds., Results: The methodology involved the fusion 3A and 2C at N- and C-terminuses of cpHaloTag, the immobilization of the fusion protein onto silica gel through bioorthogonal reaction, the morphological and functional characterization, the application in finding dual-target compounds. Expression of the fusion protein in E. coli system showed a yield of milligram level with the presence of 3A and 2C domains. Immobilization of the protein was achieved in 10 min with a reaction efficiency more than 88.5 %. Immobilized 3A-cpHalo-2C exhibited higher specificity and better retentions of canonical compounds of the two enzymes in comparison with the column containing immobilized 3A or 2C alone. In real sample application, screening analysis found that hyperoside, cymaroside, and baicalin were dual-target compounds in concert with 3A and 2C in Shuanghuanglian extract., Significance: Taking 3A and 2C as probe, we proposed a simple method for direct co-immobilization of diverse proteins from cell lysates and demonstrated an affinity chromatographic-based dual-target compound screening platform. The implications of these methodology are possible to insight the de novo design of multi-target surface for fabricating new bioanalytical methods with improved performance., 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.)

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO)., (© 2024. The Author(s).)

Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions., (© 2024. The Author(s).)

Epothilone B (Epo B), a promising antitumor compound effective against various types of cancer cells in vitro. However, its poor selectivity for tumor cells and inadequate therapeutic windows significantly limit its potential clinical application. Affibody is a class of non-immunoglobulin affinity proteins with precise targeting capability to overexpressed molecular receptors on cancer cells, has been intensively investigated due to its exceptional affinity properties. In this study, we present a targeted nanoagent self-assembled from the precursor of an affibody conjugated with Epo B via a linker containing the thioketal (tk) group that is sensitive to reactive oxygen species (ROS). The core-shell structure of the Z HER2:342 -Epo B Affibody-Drug Conjugate Nanoagent (Z-E ADCN), with the cytotoxin Epo B encapsulated within the Z HER2:342 affibody corona, leads to significantly reduced side effects on normal organs. Moreover, the abundant presence of Z HER2:342 on the surface effectively enhances the targeting capacity and tumor accumulation of the drug. Z-E ADCN can be internalized by cancer cells via HER2 receptor-mediated endocytosis followed by Epo B release in response to high levels of ROS, resulting in excellent anticancer efficacy in HER2-positive tumor models., (© 2024. The Author(s).)

Zearalenone (ZEN) is an estrogenic mycotoxin causing reproductive toxicity in livestock. Currently, lactone hydrolases are used in the enzymatic degradation of ZEN. However, most lactone hydrolases suffer from low degradation efficiency and poor thermal stability. ZHD518, as a documented neutral enzyme for ZEN degradation, exhibits high enzymatic activity under neutral conditions. In this study, a multifunctional peptide S1v1-(AEAEAHAH) 2 was fused to the N-terminus of ZHD518. Compared with the wild-type enzyme, the peptide fusion significantly enhanced protein expression by 1.28 times, enzyme activity by 9.27 times, thermal stability by 37.08 times after incubation at 45 °C for 10 min and enzyme stability during long-term storage. Moreover, ZEN concentrations in corn bran, corn germ meal, and corn gluten powder decreased from 5.29 ± 0.04, 5.31 ± 0.03, and 5.30 ± 0.01 μg/g to 0.48 ± 0.05, 0.48 ± 0.06, and 0.21 ± 0.04 μg/g, respectively, following a 60 min treatment with S1v1-GS-ZHD518, resulting in degradation rates of 90.98, 91.00, and 95.32%, respectively. In conclusion, the properties of S1v1-GS-ZHD518, such as its efficient degradability, high temperature resistance and storage resistance, offer the possibility of its application in food or feed.

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli , and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro . Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents., Importance: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro . We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection., Competing Interests: The authors declare no conflict of interest.

Proteins implement many useful functions, including binding ligands with unparalleled affinity and specificity, catalyzing stereospecific chemical reactions, and directing cell behavior. Incorporating proteins into materials has the potential to imbue devices with these desirable traits. This review highlights recent advances in creating active materials by genetically fusing a self-assembling protein to a functional protein. These fusion proteins form materials while retaining the function of interest. Key advantages of this approach include elimination of a separate functionalization step during materials synthesis, uniform and dense coverage of the material by the functional protein, and stabilization of the functional protein. This review focuses on macroscale materials and discusses (i) multiple strategies for successful protein fusion design, (ii) successes and limitations of the protein fusion approach, (iii) engineering solutions to bypass any limitations, (iv) applications of protein fusion materials, including tissue engineering, drug delivery, enzyme immobilization, electronics, and biosensing, and (v) opportunities to further develop this useful technique.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (K i ) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125 I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE., 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.)

The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule.

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.)

Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca 2+ -dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (~33.6-fold lower host cell DNA and ~6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies., (© 2024 The Protein Society.)

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 ×  1 ::CCDC124, GFP11 ×  4 ::CCDC124, or CCDC124::GFP11 ×  4 ). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 ×  1(or X4) ::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11  × 4 ::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 ×  4 ::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A DNA cassette assembly method is described which utilizes inexpensive oligomers no longer than 40 nt in length. The five-segment cassettes have 20 nt overlaps which give an effective length of 80 nt, making it possible to code for peptides up to 20 amino acids long. The cassettes have three phosphate free nicks, which can be successfully inserted into plasmid DNA and used to transform E. coli. The nicks are repaired in vivo by an unknown mechanism. Insertions are not successful for cassettes with greater than three nicks. A procedure is provided for rapid turnaround from DNA design to peptides, which are easily isolated as C-terminal fusions with GFP. The technique generally gives the expected sequence, with errors which occur about 1% of the time. Several representative DNA inserts are described which illustrate the method, as well as chemical details on the new peptides coded for. The peptides can be readily mutated to make it possible to understand how polar and aromatic residues affect GFP-fusion solubility, and how histidine residues can be strategically placed in a peptide for good IMAC retention. The method can be used to explore a large number of new designed peptides as fusion products quickly and economically., Competing Interests: The author has declared that no competing interests exist., (Copyright: © 2024 Jonathan Filley. 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.)

The Caspase-based fusion protein technology (CASPON) allows for universal cleavage of fusion tags from proteins of interest to reconstitute the native N-terminus. While the CASPON enzyme has been optimized to be promiscuous against a diversity of N-terminal peptides, the cleavage efficacy for larger proteins can be surprisingly low. We develop an efficient means to rationalize and predict the cleavage efficiency based on a structural representation of the intrinsically disordered N-terminal peptides and their putative interactions with the CASPON enzyme. The number of favorably interacting N-terminal conformations shows a very good agreement with the experimentally observed cleavage efficiency, in agreement with a conformational selection model. The method relies on computationally cheap molecular dynamics simulations to efficiently generate a diverse collection of N-terminal conformations, followed by a simple fitting procedure into the CASPON enzyme. It can be readily used to assess the CASPON cleavability a priori.

Introduction: The clinical success of mRNA vaccine during the COVID-19 pandemic has inspired emerging approaches to elevate mRNA vaccine immunogenicity. Among them, antigen fusion protein designs for improved immune cell targeting have been shown to augment humoral immunity against small antigen targets., Methods: This research demonstrates that SARS-CoV-2 receptor binding domain (RBD) fusion with a minimalistic peptide segment of complement component 3b (C3b, residues 727-767) ligand can improve mRNA vaccine immunogenicity through antigen targeting to complement receptor 1 (CR1). We affirm vaccines' antigenicity and targeting ability towards specific receptors through Western blot and immunofluorescence assay. Furthermore, mice immunization studies help the investigation of the antibody responses., Results: Using SARS-CoV-2 Omicron RBD antigen, we compare mRNA vaccine formulations expressing RBD fusion protein with mouse C3b peptide (RBD-mC3), RBD fusion protein with mouse Fc (RBD-Fc), and wild-type RBD. Our results confirm the proper antigenicity and normal functionality of RBD-mC3. Upon validating comparable antigen expression by the different vaccine formulations, receptor-targeting capability of the fusion antigens is further confirmed. In mouse immunization studies, we show that while both RBD-mC3 and RBD-Fc elevate vaccine immunogenicity, RBD-mC3 leads to more sustained RBD-specific titers over the RBD-Fc design, presumably due to reduced antigenic diversion by the minimalistic targeting ligand., Conclusion: The study demonstrates a novel C3b-based antigen design strategy for immune cell targeting and mRNA vaccine enhancement., Competing Interests: The authors declare no conflicts of interest in this research., (© 2024 Chiu et al.)

Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand-protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep-Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1-2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from Salvia miltiorrhiza extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a K D value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His 6 -tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His 6 -tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

For the study of amyloid beta (Aβ) associated toxicity which is supposed to be the main pathological agent in Alzheimer's disease (AD), it is important to secure Aβ peptide with appropriate biological activity. However, commercial and synthetic Aβ often have some pitfalls like less cell toxicity, prompt aggregation and excess price, using recombinant technology, these issues can be resolved though the method also suffered from some problems such as low yield, aggregation and prolong time to purify. Thus, we previously developed an easy, economic and convenient method for Aβ42 purification using highly expressed GroES-Ubiquitin-Aβ42 fusion protein. The method was efficient, but further development was performed to improve the procedure and increase the yield. Focus was on the isolation of the fusion protein (GroES-Ubiquitin) from Aβ42 peptide. After a series of systematic testing with several chemicals, we found that methanol could precipitate efficiently the fusion protein, while the Aβ peptide was recovered in the supernatant. By this method, Aβ peptide was easily purified without tedious chromatographic steps which are main obstacles to purify the peptide in the previous method. This method yielded ~20 mg highly pure Aβ42 peptide from 1-liter bacterial culture. Different biophysical characterizations and bioactivity assays indicate that the peptide purified using this method was competitive with others which have been previously reported whereas considering the simplicity, final yield and time of purification, this method is the optimal solution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Haque, Park. 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.)

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins., (© 2024. The Author(s).)

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Wild-type Lactococcus lactis strain LAC460 secretes prophage-encoded bacteriocin-like lysin LysL, which kills some Lactococcus strains, but has no lytic effect on the producer. LysL carries two N-terminal enzymatic active domains (EAD), and an unknown C-terminus without homology to known domains. This study aimed to determine whether the C-terminus of LysL carries a cell wall binding domain (CBD) for target specificity of LysL. The C-terminal putative CBD region of LysL was fused with His-tagged green fluorescent protein (HGFPuv). The HGFPuv&#95;CBDlysL gene fusion was ligated into the pASG-IBA4 vector, and introduced into Escherichia coli. The fusion protein was produced and purified with affinity chromatography. To analyse the binding of HGFPuv&#95;CBDLysL to Lactococcus cells, the protein was mixed with LysL-sensitive and LysL-resistant strains, including the LysL-producer LAC460, and the fluorescence of the cells was analysed. As seen in fluorescence microscope, HGFPuv&#95;CBDLysL decorated the cell surface of LysL-sensitive L. cremoris MG1614 with green fluorescence, whereas the resistant L. lactis strains LM0230 and LAC460 remained unfluorescent. The fluorescence plate reader confirmed the microscopy results detecting fluorescence only from four tested LysL-sensitive strains but not from 11 tested LysL-resistant strains. Specific binding of HGFPuv&#95;CBDLysL onto the LysL-sensitive cells but not onto the LysL-resistant strains indicates that the C-terminus of LysL contains specific CBD. In conclusion, this report presents experimental evidence of the presence of a CBD in a lactococcal phage lysin. Moreover, the inability of HGFPuv&#95;CBDLysL to bind to the LysL producer LAC460 may partly explain the host's resistance to its own prophage lysin., (© 2024. The Author(s).)

The small GTPase Ras plays an important role in intracellular signal transduction and functions as a molecular switch. In this study, we used a photoresponsive protein as the molecular regulatory device to photoregulate Ras GTPase activity. Photo zipper (PZ), a variant of the photoresponsive protein Aureochrome1 developed by Hisatomi et al. was incorporated into the C-terminus of Ras as a fusion protein. The three constructs of the Ras-PZ fusion protein had spacers of different lengths between Ras and PZ. They were designed using an Escherichia coli expression system. The Ras-PZ fusion proteins exhibited photoisomerization upon blue light irradiation and in the dark. Ras-PZ dimerized upon light irradiation. Moreover, Ras GTPase activity, which is accelerated by the Ras regulators guanine nucleotide exchange factors and GTPase-activating proteins, is controlled by photoisomerization. It has been suggested that light-responsive proteins are applicable to the photoswitching of the enzymatic activity of small GTPases as photoregulatory molecular devices., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139., Competing Interests: Declaration of competing interest No conflict of interest declared., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Generating active, pure, and monodisperse protein remains a major bottleneck for structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM). The current methodology heavily relies on overexpressing the recombinant protein fused with a histidine tag in conventional expression systems and evaluating the quality and stability of purified protein using size exclusion chromatography (SEC). This requires a large amount of protein and can be highly laborious and time consuming. Therefore, this approach is not suitable for high-throughput screening and low-expressing macromolecules, particularly eukaryotic membrane proteins. Using fluorescent proteins fused to the target protein (applicable to both soluble and membrane proteins) enables rapid and efficient screening of expression level and monodispersity of tens of unpurified constructs using fluorescence-based size exclusion chromatography (FSEC). Moreover, FSEC proves valuable for screening multiple detergents to identify the most stabilizing agent in the case of membrane proteins. Additionally, FSEC can facilitate nanodisc reconstitution by determining the optimal ratio of membrane scaffold protein (MSP), lipids, and target protein. The distinct advantages offered by FSEC indicate that fluorescent proteins can serve as a viable alternative to commonly used affinity tags for both characterization and purification purposes. In this review, I will summarize the advantages of this technique using examples from my own work. It should be noted that this article is not intended to provide an exhaustive review of all available literature, but rather to offer representative examples of FSEC applications., (© 2023 Federation of European Biochemical Societies.)

Growing concerns regarding the emergence of highly transmissible viral diseases highlight the urgent need to expand the repertoire of antiviral therapeutics. For this reason, new strategies for neutralizing and inhibiting these viruses are necessary. A promising approach involves targeting the glycans present on the surfaces of enveloped viruses. Lectins, known for their ability to recognize specific carbohydrate molecules, offer the potential for glycan-targeted antiviral strategies. Indeed, numerous studies have reported the antiviral effects of various lectins of both endogenous and exogenous origins. However, many lectins in their natural forms, are not suitable for use as antiviral therapeutics due to toxicity, other unfavorable pharmacological effects, and/or unreliable manufacturing sources. Therefore, improvements are crucial for employing lectins as effective antiviral therapeutics. A novel approach to enhance lectins' suitability as pharmaceuticals could be the generation of recombinant lectin-Fc fusion proteins, termed "lectibodies." In this review, we discuss the scientific rationale behind lectin-based antiviral strategies and explore how lectibodies could facilitate the development of new antiviral therapeutics. We will also share our perspective on the potential of these molecules to transcend their potential use as antiviral agents., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.M. is listed as an inventor on patents and patent applications concerning the composition and utility of Avaren-Fc, which is described in this manuscript. These include US patent numbers 8,802,822 and 9,133,252; PCT application numbers PCT/US 2018/017,617, PCT/US 2022/012,853, and PCT/US 2022/070,961., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications., (© 2024 Wiley Periodicals LLC.)

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.)

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine β-sheets, Cys 30 -Cys 67 , Cys 38 -Cys 58 , Cys 53 -Cys 90 , Cys 56 -Cys 66 , and Cys 81 -Cys 102 participating in the formation of five pairs of disulfide bonds. In vitro experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 μmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 μmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation., 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.)

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins., (Copyright © 2024 The Authors. Published by Elsevier Inc. 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.)

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.)

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.)

This study presents the design and fabrication of an innovative vaccine candidate targeting Pseudomonas aeruginosa (P. aeruginosa). The vaccine consists of gelatin nanoparticles (GNPs) encapsulating a chimeric protein (CP) derived from the ExoS and OprI proteins from P. aeruginosa. The physicochemical properties of the GNPs were assessed using dynamic light scattering (DLS) and electron microscopy. The toxicity, encapsulation efficacy, release profile, and effectiveness of CP-encapsulated GNPs (CP-GNPs) in an animal model were investigated. The resulting nanovaccine demonstrated uniform spherical particles with an average size of 135 nm and an encapsulation efficiency of 85 %. The release assay revealed that 23 % of the antigen was released from the CP-GNPs after 20 days. The GNPs did not exhibit any toxic effects on L929 cells in vitro. The formulation induced both systemic and mucosal antibody responses. Additionally, CP-GNPs stimulated cytokine responses, including IFN-γ, IL-4, and IL-17, indicating the induction of both humoral (Th2) and cellular (Th1) responses. The CP-encapsulated GNPs formulation effectively protected the mice lungs against experimental respiratory tract infection, reducing colony count and inflammation. These findings suggest that CP-GNPs hold promise as a potential strategy for preventing respiratory tract infections caused by P. aeruginosa. Further research is needed to explore its clinical application., Competing Interests: Declaration of competing interest The authors declare that there are no identifiable conflicting financial interests or personal relationships that could potentially have influenced the findings presented in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)