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Collagen is the most abundant protein in human and mammalian structures and is a component of the mammalian extracellular matrix (ECM). Recombinant collagen is a suitable alternative to native collagen extracted from animal tissue for various biomaterials. However, due to the limitations of the expression system, most recombinant collagens are collagen fragments and lack triple helix structures. In this study, Chinese hamster ovary (CHO) cells were used to express the full-length human type I collagen α1 chain (rhCol1α1). Moreover, Endo180 affinity chromatography and pepsin were used to purify pepsin-soluble rhCol1α1 (PSC1). The amino acid composition of PSC1 was closer to that of native human type I collagen, and PSC1 contained 9.1 % hydroxyproline. Analysis of the CD spectra and molecular weight distribution results revealed that PSC1 forms a stable triple helix structure that is resistant to pepsin hydrolysis and has some tolerance to MMP1, MMP2 and MMP8 hydrolysis. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed that PSC1 can self-assemble into fibers at a concentration of 1 mg/ml; moreover, PSC1 can promote the proliferation and migration of NIH 3T3 cells. In conclusion, our data suggest that PSC1 is a highly similar type of recombinant collagen that may have applications in biomaterials and other medical fields., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alcohol dehydrogenase I from Zymomonas mobilis (zmADH1) is a zinc-dependent oxidoreductase that catalyses the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD + /NADH as a cofactor. Efforts to express zmADH1 in Escherichia coli in a soluble form have been laden with solubility difficulties. A soluble form of recombinant zmADH1 was achieved by the addition of 1 mM zinc into media. Zinc addition facilitates the proper folding of recombinant zmADH1 and significantly reduces the formation of inclusion bodies. The yield of recombinant zmADH1 represents approximately 30 mg/1 L Luria-Bertani media. Intensified production in fermenters showed a striking difference between the specific and total activities of zmADH1 produced at different zinc concentrations. The zmADH1 showed an affinity to medium-chain alcohols, especially 1-pentanol, which could be used in new greener routes for preparation of aldehydes and alcohols., 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.)

Adiponectin (ADPN) exerts various cellular and metabolic functions by activating signaling pathways, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, the protein kinase B (Akt) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. However, generating functional recombinant human adiponectin (hADPN) in bacterial or mammalian cells is challenging. Although ADPN agonist peptides have been developed, problems like stability, solubility, and affinity for receptors remain. Recently, a genome-edited chicken bioreactor system was established, ensuring efficient ADPN production with optimal post-transcriptional modifications. We assessed the ability of egg white (EW)-derived hADPN, commercial hADPN, various ADPN agonist peptides, and globular ADPN on activation of the ERK1/2, Akt, and p38 MAPK pathways. EW-derived hADPN, abundant in hexamers and high molecular weight multimers, significantly phosphorylated ERK1/2 in serum-starved HEK293 cells after 15 min of treatment. Comparative analysis revealed that EW-derived hADPN and commercial hADPN induced greater phosphorylation of ERK1/2, Akt, and p38 MAPK than ADPN agonist peptides and globular ADPN, with EW-derived hADPN showing the highest activation. In summary, the finding that EW-derived hADPN strongly activates the ERK1/2, Akt, p38 MAPK signaling pathways highlights that an ADPN production system based on genome-edited chickens is an advantageous alternative to existing methods., 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.)

Cultivations of Chinese Hamster Ovary (CHO) cells in a perfusion setup were conducted in the presence of super physiological concentrations of L-Arginine to investigate the impact on transmission through the perfusion filter for production of a recombinant domain antibody. Our study revealed that the presence of L-Arginine within the range of 30-50 mM had a positive impact on transmission. However, the higher concentrations were found to have a negative correlation with cell viability, and an optimal concentration of approximately 40 mM was identified. The supplementation of L-Arginine improved overall cultivation performance and enhanced product quality attributes. As a result, our findings demonstrate that the supplementation of L-Arginine to mammalian perfusion cultivations stands as an effective method to address transmission issues, exerting a broad impact on process and production of recombinant proteins., Competing Interests: Declaration of Competing Interest All authors are employed by and own shares in Novo Nordisk A/S, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antibodies are powerful tools for the therapy and diagnosis of various diseases. In addition to conventional hybridoma-based screening, recombinant antibody-based screening has become a common choice; however, its application is hampered by two factors: (1) screening starts after Ig gene cloning and recombinant antibody production only, and (2) the antibody is composed of paired chains, heavy and light, commonly expressed by two independent expression vectors. Here, we introduce a method for the rapid screening of recombinant monoclonal antibodies by establishing a Golden Gate-based dual-expression vector and in-vivo expression of membrane-bound antibodies. Using this system, we demonstrate the rapid isolation of influenza cross-reactive antibodies with high affinity from immunized mice within 7 days. This system is particularly useful for isolating therapeutic or diagnostic antibodies, for example during foreseen pandemics., Competing Interests: TW, HH, YM, FY, TH, NK, TK, OO, HF No competing interests declared, (© 2024, Watanabe et al.)

Cervical cancer is the fourth most prevalent cancer among women globally, with Thai women ranking it as the third most common. At present, a prophylactic vaccine, containing virus-like particles (VLPs) of HPV L1 capsid protein, is widely recognized as one of the major prevention strategies for cervical cancer. Unfortunately, due to a low cross-protection among subtypes, protection against each HPV subtype requires vaccination with VLPs of that specific subtype. This study aimed to optimize the cultivation medium and conditions to maximize the volumetric yield of HPV52 L1 protein produced by the recombinant H. polymorpha HPV52. The results revealed that supplementation of a complex organic nitrogen source HySoy (at 10 g/L) into SYN6 medium resulted in a significantly higher specific HPV52 L1 yield and the maximum specific and volumetric HPV52 L1 protein yield were obtained at 30℃. In this study, the volumetric yield of HPV52 L1 could be increased from 50.9 mg/L in flask-scale cultivation to 2728 mg/L in High Cell Density Cultivation or HCDC (53-folds) with a significant improvement in terms of productivity, from 0.85 mg/L.h in flask-scale cultivation to 22.7 mg/L.h in HCDC (27-folds)., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Electrocatalytic carbon dioxide (CO 2 ) reduction by CO 2 reductases is a promising approach for biomanufacturing. Among all known biological or chemical catalysts, hydrogen-dependent carbon dioxide reductase from Thermoanaerobacter kivui (TkHDCR) possesses the highest activity toward CO 2 reduction. Herein, we engineer TkHDCR to generate an electro-responsive carbon dioxide reductase considering the safety and convenience. To achieve this purpose, a recombinant Escherichia coli TkHDCR overexpression system is established. The formate dehydrogenase is obtained via subunit truncation and rational design, which enables direct electron transfer (DET)-type bioelectrocatalysis with a near-zero overpotential. By applying a constant voltage of -500 mV (vs. SHE) to a mediated electrolytic cell, 22.8 ± 1.6 mM formate is synthesized in 16 h with an average production rate of 7.1 ± 0.5 μmol h -1 cm -2 , a Faradaic efficiency of 98.9% and a half-cell energy efficiency of 94.4%. This study provides an enzyme candidate for high efficient CO 2 reduction and opens up a way to develop paradigm for CO 2 -based bio-manufacturing., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Vision and magnetoreception in navigating songbirds are strongly connected as recent findings link a light dependent radical-pair mechanism in cryptochrome proteins to signalling pathways in cone photoreceptor cells. A previous yeast-two-hybrid screening approach identified six putative candidate proteins showing binding to cryptochrome type 4a. So far, only the interaction of the cone specific G-protein transducin α-subunit was investigated in more detail. In the present study, we compare the binding features of the G-protein α-subunit with those of another candidate from the yeast-two-hybrid screen, cellular retinol binding protein. Purified recombinant European robin retinol binding protein bound retinol with high affinity, displaying an EC 50 of less than 5 nM, thereby demonstrating its functional state. We applied surface plasmon resonance and a Förster resonance transfer analysis to test for interactions between retinol binding protein and cryptochrome 4a. In the absence of retinol, we observed no robust binding events, which contrasts the strong interaction we observed between cryptochrome 4a and the G-protein α-subunit. We conclude that retinol binding protein is unlikely to be involved in the primary magnetosensory signalling cascade., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a glycosylphosphatidylinositol (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoite/sporoblast stages, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequence identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic, with a low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.6%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and -10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2., 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.)

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).)

The production of therapeutic glycoproteins is primarily expensive due to the necessity of culturing mammalian cells. These systems often require complex and costly culture media and typically yield low amounts of protein. Leishmania tarentolae, a non-pathogenic protozoan to mammals, has emerged as a cost-effective alternative system for heterologous glycoprotein expression due to its suitability for large-scale production using low-cost culture media, and its ability to perform mammalian-like post-translational modifications, including glycosylation. Nevertheless, differences in the carbohydrate residues at the end of N-glycan chains are observed in Leishmania compared to mammalian cells due to the absence of biosynthetic enzymes in Leishmania that are required for the incorporation of terminal sialic acid. In this study, a genetically optimized L. tarentolae cell line was engineered for the production of recombinant interferon-β (IFN-β) featuring a complete mammalian N-glycosylation profile. Genomic and metabolomic analyses revealed that heterologous expression of the sialyltransferase enzyme and cultivation in a medium containing sialic acid were sufficient to generate mammalian-like protein N-glycosylation. N-glycan mass spectrometry analysis demonstrated a glycosylation pattern compatible with the incorporation of sialic acid into the glycan structure. In vitro IFN-β activity indicated that the expressed protein exhibited reduced inflammatory effects compared to IFN-beta produced by other platforms, such as bacteria, non-optimized L. tarentolae, and mammalian cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of a potent endogenous calcification inhibitor, plasma inorganic pyrophosphate (PPi); the deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1 that generates PPi and is now in clinical trials, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of Abcc6 -/- mice. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6 -/- mice. When Abcc6 -/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 h. When Abcc6 -/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising second generation enzyme therapy for PXE, the first generation of which is currently in clinical testing., Competing Interests: Declaration of interests D.T.B. and P.R.S. are inventors on patents owned by Yale University for therapeutics treating ENPP1 deficiency. D.T.B. is an equity holder and receives research and consulting support from Inozyme Pharma, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P T5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

Heat-shock protein 47 (HSP47) is a potential target for inhibitors that ameliorate fibrosis by reducing collagen assembly. In an effort to develop a structure-based drug-design system, it was not possible to replicate a previous literature result (PDB entry 4au4) for apo dog HSP47; instead, crystal forms were obtained in which pairs of dog HSP47 molecules interacted through a noncleavable C-terminal His-tag to build up tetramers, all of which had multiple molecules of HSP47 in the asymmetric unit and none of which diffracted as well as the literature precedent. To overcome these difficulties, a two-pronged approach was followed: (i) the His-tag was moved from the C-terminus to the N-terminus and was made cleavable, and (ii) Adnectin (derived from the tenth domain of human fibronectin type III) crystallization chaperones were developed. Both approaches provided well diffracting crystals, but the latter approach yielded crystal forms with only one or two HSP47 complexes per asymmetric unit, which made model building less onerous.

Mammalian prions are neurotropic pathogens formed from PrP Sc assemblies, a misfolded variant of the host-encoded prion protein PrP C . Multiple PrP Sc conformations or strains self-propagate in host populations or mouse models of prion diseases, exhibiting distinct biological and biochemical phenotypes. Constrained interactions between PrP Sc and PrP C conformations confer species specificity and regulate cross-species transmission. The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant PrP (rPrP) has been instrumental in demonstrating the protein-only nature of prions. Yet, their ability to encode different strains and transmit between species remains poorly studied, hampering their use in exploring structure-to-strain relationships. Fibrillar assemblies from rPrP with hamster, mouse, human, and bovine primary structures were generated and tested for transmission and adaptation in tg7 transgenic mice expressing hamster PrP C . All assemblies, except the bovine ones, were fully pathogenic on the primary passage, causing clinical disease, PrP Sc brain deposition, and spongiform degeneration. They exhibited divergent adaptation processes and strain properties upon subsequent passage. Assemblies of hamster origin propagated without apparent need for adaptation, those of mouse origin adapted abruptly, and those of human origin required serial passages for optimal fitness. Molecular analyses revealed the presence of endogenously truncated PrP Sc species in the resulting synthetic strains that lack the 90-140 amino acid region considered crucial for infectivity. In conclusion, rPrP assemblies provide a facile means of generating novel prion strains with adaptative/evolutive properties mimicking genuine prions. The PrP amino acid backbone is sufficient to encode different strains with specific adaptative properties, offering insights into prion transmission and strain diversity., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Å resolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central β-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. In this study, the crystal structure of ApePCNA1, one of three PCNAs from the thermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols., (open access.)

The aim of this study was to investigate the catalytic activity of 26 Cytochrome P450 3A4 (CYP3A4) variants and drug interactions on imatinib metabolism in recombinant insect microsomes. This study was designed with an appropriate incubation system and carried out in the constant temperature water. By using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to measure the quantities of its metabolite N-desmethyl imatinib, to elucidate the impacts of the CYP3A4 genetic polymorphism and drug interactions on the metabolism of imatinib. Consequently, as compared to CYP3A4.1, the intrinsic clearance (CL int ) values of the variations were dramatically changed, rising from 2.34 % to 120.57 %. CYP3A4.14 showed an increase in CL int in comparison to CYP3A4.1, and the remaining 24 variants demonstrated decreases in catalytic activity for the metabolism of imatinib. In addition, the metabolism of imatinib was decreased to varied degrees by ketoconazole, itraconazole, and fluconazole in CYP3A4.1 and CYP3A4.18. Moreover, most of CYP3A4 variants showed similar trend of enzyme activity under different substrates of imatinib and cabozantinib, except 6 variants (CYP3A4.3,.4,.10,.15,.29 and.31). The first study of the effects of 26 CYP3A4 variants on imatinib metabolism will contribute to the clinical evaluation of imatinib and help personalize therapy in clinical settings., 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 Masson SAS.. All rights reserved.)

The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 μmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 μmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 μmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Current medical countermeasures (MCMs) for nerve agent poisoning have limited efficacy, and can cause serious adverse effects, prompting the requirement for new broad-spectrum therapeutics. Human plasma-derived butyrylcholinseterase (huBChE) is a promising novel bioscavenger MCM which has shown potential in animal studies, however, is economically prohibitive to manufacture at scale. This study addresses current challenges for the economical production of a bioactive and long-acting recombinant huBChE (rBChE) in mammalian cells by being the first to directly compare novel rBChE design strategies. These include co-expression of a proline rich attachment domain (PRAD) and fusion of BChE with a protein partner. Additionally, a pre-purification screening method developed in this study enables parallel comparison of the expression efficiency, activity and broad-spectrum binding to nerve agents for ten novel rBChE molecular designs. All designed rBChE demonstrated functionality to act as broad-spectrum MCMs to G, V and A series nerve agents. Expression using the ExpiCHO™ Max protocol provided greatest expression levels and activity for all constructs, with most rBChE expressing poorly in Expi293™. Fc- or hSA-fused rBChE significantly outperformed constructs designed to mimic huBChE, including PRAD-BChE, and proved an effective strategy to significantly improve enzyme activity and expression. Choice of protein partner, directionality and the addition of a linker also impacted fusion rBChE activity and expression. Overall, hSA fused rBChE provided greatest expression yield and activity, with BChE-hSA the best performing construct. The purified and characterised BChE-hSA demonstrated similar functionality to huBChE to be inhibited by GD, VX and A-234, supporting the findings of the pre-screening study and validating its capacity to assess and streamline the selection process for rBChE constructs in a cost-effective manner. Collectively, these outcomes contribute to risk mitigation in early-stage development, providing a systematic method to compare rBChE designs and a focus for future 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., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post-translational modifications like glycosylation of these Chlamydomonas-made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild-type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild-type strain demonstrated that the three N-glycosylation sites are 100% glycosylated with mature N-glycans containing four to five mannose residues and carrying core xylose, core fucose and O-methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N-linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N-glycosylation of the Chlamydomonas-made biologics., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Cold-active lipase from the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania) was cloned and characterized as an extremophilic biocatalyst for silybin acylation. Structural analyses highlighted conserved motifs confirming a functional lipase and the presence of primary structure elements for catalysis at low temperatures. The recombinant enzyme (PSL2) heterologously expressed in Escherichia coli was purified in one step by affinity chromatography with a yield of 12.08 ± 1.72 µg L -1 of culture and a specific activity of 20.1 ± 3.2 U mg -1 at 25 °C. Functional characterization of PSL2 showed a neutral (7.2) optimal pH and a high thermal stability up to 90 °C. Also, this lipase was stable in the presence of different organic solvents, with 60% residual activity when using 20% DMSO. Kinetic measurements indicated performant catalytic efficiency of PSL2 for different short and long chain fatty acids, with Km in the mM range. The catalytic activity of PSL2 was assessed for silybin acylation with various fatty acids and fatty acid methyl esters, demonstrating a 90% silybin conversion when methyl decanoate ester was used. This result clearly highlights the biocatalytic capability of this new cold-active lipase.

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite., 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.)

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene ( lip 4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application.

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in Nicotiana benthamiana leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins.

Since microalgae face various environmental stresses for the high production of biofuels, multiple studies have been performed to determine if microalgae are resistant to these various stresses. In this study, the viability of cells under various abiotic stresses was investigated by introducing a putative germin-like protein (GLP) from Neopyropia yezoensis , which was known to be related in the resistance to abiotic stresses. The expression of GLP in Chlamydomonas reinhardtii allowed cells to grow better in various abiotic stress environments. In nitrogen starvation conditions, recombinant cells accumulated the lipid droplet 1.46-fold more than wild-type cells and responded more rapidly to form palmelloid forms. Under high-temperature, hydrogen peroxide conditions and saline stress, the survival rate was increased 3.5 times, 2.19 times, and 3.19 times in recombinant C. reinhardtii with GLP, respectively. The expression level of genes related to pathways in response to various stresses increased 2-fold more under those conditions. This result will be useful for the development of microalgae that can grow better and produce more biofuels under different stress conditions.

The deposition of amyloid β-protein (Aβ) in the brain is the main pathogenesis of Alzheimer's disease (AD). The development of potent inhibitors against Aβ aggregation is one of the effective strategies to combat AD. Endogenous transthyretin (TTR) can inhibit Aβ fibrillization via hydrophobic interactions, but its weak inhibitory potency hinders its application in AD therapy. Here, different recombinant TTRs were designed by cationic surface charge engineering. Compared with TTR, all positively charged recombinant TTRs showed enhanced capability in inhibiting Aβ aggregation, especially the recombinant protein obtained by mutating the acidic amino acid in TTR to arginine (TTR-nR) exhibited excellent inhibitory effect. Among them, TTR-7R remarkably increased the inhibitory potency against Aβ, which could effectively inhibit Aβ 40 fibrillization at a very low concentration (0.5 μM). In addition, TTR-7R increased cultured cell viability from 62% to 89%, scavenged amyloid plaques in AD nematodes, and prolonged nematode lifespan by 5 d at 2 μM. Thermodynamic studies demonstrated that TTR-7R, enriching in positive charges, presented hydrophobic interactions and enhanced electrostatic interactions with Aβ 40 , leading to a significantly enhanced inhibitory capacity of TTR-7R. The research provided insights into the development of efficient recombinant protein inhibitors for AD treatment.

Chinese hamster ovary (CHO) cells are common mammalian cell lines for expressing recombinant proteins, yet the expression level of recombinant proteins is still hindered. Vector optimization and cell line modification are the key factors to improve the expression of recombinant proteins. In this study, the vector was optimized by adding the regulatory elements Kozak and Leader to the upstream of target gene to detect the transient and stable expression of recombinant proteins. Results indicated that the expression level of target proteins with the addition of regulatory elements was significantly increased compared with the control group. In addition, the inhibition of apoptotic pathway has great potential to increase recombinant protein production, and Apaf1 protein dependent on the mitochondrial apoptosis pathway plays an important role in this respect. The knockout of apoptotic gene Apaf1 in CHO cells can also increase recombinant protein production. Therefore, the vector was optimized by adding regulatory elements, and the cell line was modified by using CRISPR/Cas9 technology to establish a novel CHO cell expression system, which remarkably improved the expression level of recombinant proteins and laid the foundation for the large-scale production of recombinant proteins., (© 2024. The Author(s).)

Background: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom., Results: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system., Conclusions: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications., (© 2024. The Author(s).)

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems., (© 2024. The Author(s).)

Salmonella Enteritidis is a major foodborne pathogen throughout the world and the increase in antibiotic resistance of Salmonella poses a significant threat to public safety. Natural nanobodies exhibit high affinity, thermal stability, ease of production, and notably higher diversity, making them widely applicable for the treatment of viral and bacterial infections. Recombinant expression using Lactococcus lactis leverages both acid resistance and mucosal colonization properties of these bacteria, allowing the effective expression of exogenous proteins for therapeutic effects. In this study, nine specific nanobodies against the flagellar protein FliC were identified and expressed. In vitro experiments demonstrated that FliC-Nb-76 effectively inhibited the motility of S. Enteritidis and inhibited its adhesion to and invasion of HIEC-6, RAW264.7, and chicken intestinal epithelial cells. Additionally, a recombinant L. lactis strain secreting the nanobody, L. lactis-Nb76, was obtained. Animal experiments confirmed that it could significantly reduce the mortality rates of chickens infected with S. Enteritidis, together with alleviating the inflammatory response caused by the pathogen. These results provide a novel strategy for the treatment of antibiotic-resistant S. Enteritidis infection in the intestinal tract., (© 2024. The Author(s).)

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies., (© 2024. The Author(s).)

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli , purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Background: Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome., Results: The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response., Conclusions: These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications., (© 2024. The Author(s).)

This study aimed to increase the antifungal and insecticidal activities of NaD1, as an antimicrobial peptides (AMP), by improving its interaction with the fungal cell wall and chitin monomeric units in insect midguts. Hence, the chitin-binding domains (CBDs) of wheat germ agglutinin protein (WGA) were fused to either N- or C-terminus of NaD1 generating transgenic Nicotiana tabacum hairy roots (HRs). Molecular assessments confirmed the integration of NaD1 transgenes, their transcription and production of recombinant peptides in the HR lines. Total protein of (CBD) 4 -NaD1 and NaD1-(CBD) 4 transgenic lines inhibited the growth of Pyricularia oryzae mycelium, suggesting that fusion of CBD to NaD1 can increase NaD1 half-life, leading to higher affinity toward cell wall chitin. Furthermore, feeding the third-instar larvae of Chilo suppressalis with both (CBD) 4 -NaD1 and NaD1-(CBD) 4 extracts exhibited a higher mortality rate. Both NaD1-CBDs caused a significant decrease in trypsin (TRY) and chymotrypsin (CTR) activities in the larvae, while enhancing the activity of antioxidant enzymes CAT, POD, APX, and SOD. Therefore, feeding the larvae by total extract of NaD1-(CBD) 4 and (CBD) 4 -NaD1 HR lines probably increased affinity to midgut chitin in C. suppressalis, enhancing insecticidal activities. Overall, the results indicate that recombinant peptides are effective in enhancing fungal and insect resistance., (© 2024. The Author(s).)

Background: The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low K M value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly., Results: Firstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkat oNPGal /L culture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkat oNPGal /g DCW /h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkat oNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter)., Conclusion: This study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme., (© 2024. The Author(s).)

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni 2+ -NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS., (© 2024. The Author(s).)

Background: Timothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings., Objective: The main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip., Methods: Recombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed., Results: Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort., Conclusions: Recombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies., (© 2024. The Author(s).)

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis., (© 2024. The Author(s).)

The α-amylase BmaN1 from Bacillus megaterium NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in Escherichia coli ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from B. megaterium NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements., Competing Interests: The authors declare no conflict of interest.

Background: The potency of inactivated and recombinant influenza vaccines is measured using the single-radial immunodiffusion (SRID) assay. The strain-specific antigen and antibody potency reagents required for the assay are prepared and distributed by regulatory agencies to ensure vaccine standardization, but timely reagent production is always challenging. This poses unique concerns for rapid pandemic responses. Alternative methods have been described for generating strain-specific potency antibody reagents without the need for live influenza virus, but such methods are infrequently used, suggesting the need for additional antigen expression approaches., Methods: We describe a rapid process using a mammalian expression system to produce recombinant influenza hemagglutinin (rHA). This platform was used to generate rHA from two H5 clade 2.3.4.4 influenza viruses, in both soluble ectodomain or full-length HA forms, and a soluble ectodomain rHA from an influenza H2 virus., Results: The purified rHAs were used as immunogens to produce HA antibody reagents that were tested for suitability in the SRID assay to accurately measure the potency of inactivated pandemic influenza vaccines. Antibody reagents generated to either ectodomain or full-length rHA worked well in the SRID assay and resulted in vaccine potency values equivalent to those generated with standard reference antibodies., Conclusions: The results demonstrate that rHA produced from a simple mammalian cell transfection method can be used to generate HA antibody suitable for use in the influenza vaccine SRID potency assay and suggest a practical means by which an extensive library of pandemic reagents can easily be prepared in advance of and during an influenza emergency., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation. The γL96Y mutation presented the best profile of oxidative protection unaccompanied by loss of protein stability and function. N-terminal deletions were constructed to facilitate the production of recombinant hemoglobin by fermentation and phenylalanine insertions in the heme pocket to decrease the rate of NO dioxygenation. The resultant mutant (αV1del. αL29F, γG1del. γV67F, γL96Y) significantly decreased NO scavenging and lipid peroxidation in vitro. Unlike native hemoglobin or a recombinant control (αV1del, γG1del), this mutation showed no increase in blood pressure immediately following infusion in a rat model of reperfusion injury, suggesting that it was also able to prevent NO scavenging in vivo. Infusion of the mutant also resulted in no meaningful adverse physiological effects apart from diuresis, and no increase in oxidative stress, as measured by urinary isoprostane levels. Following PEGylation via the Euro-PEG-Hb method to increase vascular retention, this novel protein construct was compared with saline in a severe rat reperfusion injury model (45% blood volume removal for 90 minutes followed by reinfusion to twice the volume of shed blood). Blood pressure and survival were followed for 4 h post-reperfusion. While there was no difference in blood pressure, the PEGylated Hb mutant significantly increased survival., (© 2024. The Author(s).)

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X-ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Å resolution using an in-house X-ray diffractometer and belonged to an orthorhombic space group, with unit-cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (V M ) was calculated to be 2.18 Å 3  Da -1 , suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing.

The detection of pathogenicity and immunogenicity in Vibrio parahaemolyticus poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. V. parahaemolyticus synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by VP_RS08405 in V. parahaemolyticus was identified. Based on sequence alignment analysis, V. parahaemolyticus VP_RS08405 has high homology to E. coli lpxL, lpxM and lpxP which encode the three secondary acyltransferases of lipid A. Therefore, V. parahaemolyticus VP_RS08405 was cloned into pBAD33, and the resulting pB08405 was introduced in E. coli mutants WHL00 in which lpxL was deleted, WHM00 in which lpxM was deleted, WHP00 in which lpxP was deleted, and WH300 in which lpxL, lpxM and lpxP were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2'-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, VP_RS08405 was overexpressed in E. coli mutants WH001 in which waaA was deleted, and WH400 in which waaA, lpxL, lpxM and lpxP were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in V. parahaemolyticus., (Copyright © 2024 Elsevier Inc. All rights reserved.)

(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme dihydrodaidzein reductase in (S)-equol synthesis. After iterative combinatorial mutagenesis, an effective mutant S118G/T169A capable of significantly increasing (S)-equol yield was obtained. Computational analyses illustrated that the main reason of the increased activity relied on the decreased critical distance and more stable interacting conformation. Then, the reaction optimization was performed, and the recombinant Escherichia coli whole-cell biocatalyst harboring S118G/T169A enabled the efficient conversion of 2 mM daidzein to (S)-equol, achieving conversion rate of 84.5 %, which was 2.9 times higher than that of the parental strain expressing wide type dihydrodaidzein reductase. This study provides an effective idea and a feasible method for enzyme modification and whole-cell catalytic synthesis of (S)-equol, and will greatly accelerate the process of industrial production., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 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.)

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined., Competing Interests: Declaration of competing interest All authors declare that they have no competing financial or personal interests influencing the work reported in this review paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)