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Background: Endothelial hyperpermeability-induced vascular dysfunction is a prevalent and significant characteristic in critical illnesses such as sepsis and other conditions marked by acute systemic inflammation. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and Tie2 serve as transmembrane receptors within endothelial cells (ECs), playing pivotal roles not only in maintaining EC-EC junctions but also in influencing vasculogenesis, vessel homeostasis, and vascular remodeling., Objectives: At present, the molecular basis of the PECAM-1-Tie2 interaction remains inadequately elucidated. In the study, recombinant soluble PECAM-1 (sPECAM-1) and Tie2 (sTie2) were expressed by Drosophila S2 and HEK293 expression systems, respectively. The interactions between sPECAM-1 and sTie2 were investigated using the Surface Plasmon Resonance (SPR) and size-exclusion chromatography methods. An immunofluorescence assay was used to detect the binding of sPECAM-1 and sTie2 on endothelial cells., Results: PECAM-1 was found to bind with sTie2 in a sodium and pH-dependent manner as confirmed by the ELISA, the D5-D6 domains of PECAM-1 might play a crucial role in binding with sTie2. Surface Plasmon Resonance (SPR) results showed that the full length of sPECAM-1 has the strongest binding affinity (K D  = 48.4 nM) with sTie2, compared to sPECAM-1-D1-D4 and sPECAM-1-D1-D2. This result is consistent with that in the ELISA. In addition, size-exclusion chromatography demonstrated that sPECAM-1, sTie2, and Ang1 can form a ternary complex., Conclusion: In this study, we determined that sPECAM-1 binds to sTie2 in a pH and sodium-dependent manner. The full length of sPECAM-1 has the strongest binding affinity, and the D5-D6 domains in sPECAM-1 play a crucial role in the interaction between sPECAM-1 and sTie2., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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).)

Cellulase is a complex enzyme system composed of multiple hydrolytic enzymes. It can degrade cellulose into glucose and improve the utilization efficiency of cellulose resources. Cellulase produced by microorganisms is the main method used in industry, offering the advantages of convenience and being environmentally friendly. A strain Xh-16 with high cellulase production was screened from the rotten rice straw. It was identified as Bacillus subtilis by morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Strain Xh-16 was used to degrade rice straw. After a 48 h cellulase treatment, the complete degradation and structural breakdown of the straw were observed. We cloned the endoglucanase Cel5L gene of Bacillus subtilis Xh-16 and induced expression of the cloned gene in Escherichia coli BL21 (DE3). The results showed that the coding length of Cel5L gene was 1500 bp, and the molecular weight of the encoded protein was about 55 kDa. The molecular formula is C 2456 H 3811 N 671 O 761 S 10 and it has 7709 atoms and 499 amino acids. Cel5L differs significantly from some the glycoside hydrolase family 5 cellulases because it has only one carbohydrate binding module family 3 at the C-terminus of its catalytic domain. The cellulase gene Cel5L in Xh-16 can encode active cellulase and can be heterologously expressed in Escherichia coli, which makes Escherichia coli have cellulase function. This study serves as a foundation for further research on cellulase diversity in Bacillus subtilis and offers insights for enhancing cellulase production., Competing Interests: Declarations Conflict of interest All authors have no conflicts of interest. Ethical Approval This study did not require ethics approval. Consent to Participate Not applicable. Consent for Publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Entomopathogenic fungi excrete a group of proteins to assimilate nutrients and defeat the host immune defense. Functional secretory signal sequences are needed for efficient secretion of the virulence-related proteins in recombinant strain. In this study, secretome analysis was used to explore the secreted proteins of Beauveria bassiana. Enrichment analysis indicated that B. bassiana secretome was mainly associated with metabolism of glucoside, polysaccharide, extracellular ester compound, and so on. In addition, proteins associated with biogenesis of cellular components were also enriched, including those involved in biogenesis of cell wall and vacuole. Then, four secretory signal sequences were functionally verified with green fluorescent protein as reporter. Finally, a signal sequence was used to excrete three insect venom protein serpins in B. bassiana, in which over-expression of serpin 8 gene resulted in a significant increase in fungal virulence. This study highlights that functional secretory signal sequences are potential molecular elements useful in excretion of virulence-related proteins in insect pathogenic fungi., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Trichoderma longibrachiatum UN32 is an industrially important fungus capable of producing Dendrobine-Type Total alkaloids (DTTAs). Several reports have pointed out that branched-chain amino acid aminotransferases (BCATs) participate in backbone modification or promote the production of secondary metabolites. We previously proposed that cobalt chloride increased DTTAs production in T. longibrachiatum UN32, which was associated with enhanced expression of the gene BCAT, TlBCAT (Genbank accession No. PP465542). Following cloning and characterization, the cDNA of TlBCAT was found to consists of 1191 bp, encoding a protein of 397 amino acid residues. The molecular mass of TlBCAT was about 42 kDa through SDS-PAGE analysis. The predicted pI value was 5.54. Recombinant TlBCAT can catalyze L-leu with a catalytic efficiency of 15.91 mM - 1 S - 1 . In the binding pocket, residues interacting with PLP, including Tyr68, Arg93, Tyr136, and Lys194, are highly conserved. TlBCAT exhibits a broad spectrum of substrate specificity, typical for catalyzing the transamination reaction of various branched-chain and hydrophobic amino acids, with α-ketoglutarate as the amino acceptor. Exogenous branched-chain amino acids (BCAAs) positively affect Trichoderma growth and DTTAs production. These findings offer insights into the physiological significance of BCAAs and present a novel approach for enhancing DTTAs production in Trichoderma mycelium cultures., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Escherichia coli is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic E. coli strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of lpp (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.

Precise and timely diagnosis is essential to prevent severe outcomes of leptospirosis in humans and animals. Existing diagnostic methods face challenges and limitations, underscoring the need for novel, field-applicable screening, and diagnostic tests/assays. This study evaluates the diagnostic utility of a recombinant ErpY-like lipoprotein (rErpY-LIC11966) in a latex agglutination test (LAT) for diagnosis of animal leptospirosis. The ErpY gene sequence from Leptospira interrogans serovar Pomona, excluding the signal peptide, was amplified, cloned into the pETite vector, and expressed in Escherichia coli. The expressed rErpY (∼16 kDa) was characterized by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western blot using Leptospira-specific standard sera. To assess the diagnostic potential of rErpY, Ni-NTA affinity-purified protein was used to sensitize latex-coated beads (0.8 µm colour beads), which were then employed in the LAT for standardization and optimization with standard positive and negative sera. For evaluation, the rErpY-LAT was tested on serum samples from 177 suspected animal cases and compared to the microscopic agglutination test. It showed a relative diagnostic sensitivity of 90.6%, a specificity of 89.1%, and an overall accuracy of 90%. This study proposes rErpY-LAT as a field testing/screening diagnostic tool for preliminary serodiagnosis of leptospirosis, highlighting the potential of recombinant protein-based assays to address current diagnostic challenges., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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.

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. KEY POINTS: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins., (© 2024. The Author(s).)

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca 2+ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca 2+ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37-G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233-425, glutamine-substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A 2 stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis . Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

Antivenoms are essential in the treatment of the neurotoxicity caused by elapid snakebites. However, there are elapid neurotoxins, e.g., long-chain α-neurotoxins (also known as long-chain three-finger toxins) that are barely neutralized by commercial elapid antivenoms; so, recombinant elapid neurotoxins could be an alternative or complements for improving antibody production against the lethal long-chain α-neurotoxins from elapid venoms. This work communicates the expression of a recombinant long-chain α-neurotoxin, named HisrLcNTx or rLcNTx, which based on the most lethal long-chain α-neurotoxins reported, was constructed de novo. The gene of rLcNTx was synthesized and introduced into the expression vector pQE30, which contains a proteolytic cleavage region for exscinding the mature protein, and His residues in tandem for affinity purification. The cloned pQE30/rLcNTx was transfected into Escherichia coli Origami cells to express rLcNTx. After expression, it was found in inclusion bodies, and folded in multiple Cys-Cys structural isoforms. To observe the capability of those isoforms to generate antibodies against native long-chain α-neurotoxins, groups of rabbits were immunized with different cocktails of Cys-Cys rLcNTx isoforms. In vitro, and in vivo analyses revealed that rabbit antibodies raised against different rLcNTx Cys-Cys isoforms were able to recognize pure native long-chain α-neurotoxins and their elapid venoms, but they were unable to neutralize bungarotoxin, a classical long-chain α-neurotoxin, and other elapid venoms. The rLcNTx Cys-Cys isoform 2 was the immunogen that produced the best neutralizing antibodies in rabbits. Yet to neutralize the elapid venoms from the black mamba Dendroaspis polylepis, and the coral shield cobra Aspidelaps lubricus, it was required to use two types of antibodies, the ones produced using rLcNTx Cys-Cys isoform 2 and antibodies produced using short-chain α-neurotoxins. Expression of recombinant elapid neurotoxins as immunogens could be an alternative to improve elapid antivenoms; nevertheless, recombinant elapid neurotoxins must be well-folded to be used as immunogens for obtaining neutralizing antibodies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To enhance the photoconversion performance of biphotochromic moxSAASoti protein, a substitution F97 M was introduced. In addition to enhancing the target properties, this substitution also resulted in the crystallization of the recombinant protein within living HeLa cells (also referred to as in cellulo crystallization). The phenomenon of protein crystallization in living cells is not unique, yet the mechanisms and application of in cellulo crystallization remain significant for further research. However, in cellulo crystallization is atypical for fluorescent proteins and detrimental for their biotechnological application. The objective of this study was to elucidate the underlying mechanisms responsible for the crystallization of moxSAASoti F97M in cellulo. For this purpose, the crystal structure of the green form of biphotochromic protein moxSAASoti F97M was determined at high resolution, which surprisingly has a space group, different from those of parent mSAASoti C21N . The analysis provided allowed to propose a mechanism of new crystal contacts formation, which might be a cause of in cellulo protein crystallization., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as β-glucosidase. This study involved cloning a β-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-β-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against β-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both β-glucosidase and β-glucanase activities, a characteristic rarely found in β-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing β-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify β-glucan and ferment it to produce ethanol. This gene, with its dual β-glucosidase and β-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Secreted protein Reelin is implicated in neuropsychiatric disorders and its supplementation ameliorates neurological symptoms in mouse disease models. Recombinant human Reelin protein may be useful for the treatment of human diseases, but its properties remain uncharacterized. Here, we report that full-length human Reelin was well secreted from transfected cells and was able to induce Dab1 phosphorylation. Unexpectedly, the central fragment of human Reelin was much less secreted than that of mouse Reelin. Three residues in the sixth Reelin repeat contributed to the secretion inefficiency, and their substitutions with mouse residues increased the secretion without affecting its biological activity. Our findings help efficient production of human Reelin protein for the supplementation therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Molecular cloning and functional characterization of Nemopilema nomurai venom metalloproteinases have provided deeper insights into the pathogenesis of jellyfish dermatitis. This study reports a new cDNA clone from N. nomurai tentacle venom (Transcript sequence: ID469) encoding 362 amino acid residues, belonged to astacin family and capable of disrupting the cell matrix. The N. nomurai metalloproteinase 469 (NnM469) comprises a signal peptide and propeptide, followed by metalloproteinase domain containing a zinc-binding motif, and two ShKT domains. Notably, NnM469 features a zinc-binding motif (HEXXH) at the active site, within an extended sequence of HEXXHXXGFXHE, which is unique to astacin. Immunocytochemistry revealed that NnM469 is located in the stab tube and envelope of jellyfish nematocysts. Western blot and LC-MS/MS analysis confirmed that the NnM469 protein was successfully expressed using the Pichia pastoris expression system. The recombinant NnM469 could degrade the cell matrix, resulting in the death of HaCaT cells with an IC 50 of 26.34 μg/mL. Finally, I-TASSER-generated structure and function predictions indicated that conserved Asp 53 , His 168 , His 172 , His 178 , and Tyr 227 serve as key amino acid residues for the Zn 2+ ion binding in the catalytic center. In summary, the study of the molecular characteristics and function of NnM469 presents an opportunity to develop therapeutic interventions for jellyfish venom-induced dermatitis., 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.)

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.)

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cysteine-rich peptide that plays a crucial role in the innate immune system of fish. To investigate the molecular function of LEAP-2 from olive flounder, Paralichthys olivaceus, we cloned the gene encoding LEAP-2 using PCR and expressed it in Escherichia coli. Analysis of LEAP-2 expression revealed predominant transcripts in the liver and lower levels in the intestine of olive flounder, whereas their expression levels in the liver and head kidney increased, during the initial stage of infection with the aquapathogenic bacterium Edwardsiella piscicida. Recombinant LEAP-2 (rOfLEAP-2) purified from E. coli exhibited antimicrobial activity, as demonstrated by the ultrasensitive radial diffusion assay, against both Gram-positive (Bacillus subtilis, Streptococcus parauberis, and Lactococcus garvieae) and Gram-negative (Vibrio harveyi and E. coli) bacteria, with minimum inhibitory concentrations ranging from 25 to 100 μg/mL depending on the species tested. The antibacterial activity of rOfLEAP-2 was attributed to its ability to disrupt bacterial membranes, validated by the N-phenylnaphthalen-1-amine uptake assays and scanning electron microscope analysis against E. coli, V. harveyi, B. subtilis, and L. garvieae treated with rOfLEAP-2. Furthermore, a synergistic enhancement of antibacterial activity was observed when rOfLEAP-2 was combined with ampicillin or synthetic LEAP-1 peptide, suggesting a distinct mechanism of action from those of other antimicrobial agents. These findings provide evidence for the antibacterial efficacy of LEAP-2 from olive flounder, highlighting its potential therapeutic application against pathogenic bacteria., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The K m and V max for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min -1 mg -1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy-to-use detection methods for SARS-CoV-2 variants are critical for effective epidemic prevention and control. The N protein of SARS-CoV-2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS-CoV-2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2-C11, 5E2-E8, 6C3-D8, 7C8, 9F2-E9, 12H5-D11, 13G2-C10, 14E9-F6, and 15H3-E10. Using these antibodies, we established a sandwich ELISA with 6C3-D8 as the capture antibody and 5F2-C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross-reactivity with MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E. Furthermore, this method successfully detected both wild-type SARS-CoV-2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad-spectrum applicability, providing a robust tool for detecting SARS-CoV-2 variants., (© 2024 The Societies and John Wiley & Sons Australia, Ltd.)

Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N'-diacetyl-β-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N',N″-triacetyl-β-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Beta-defensins, such as β-defensin 123 (DEFB123), are vital components of the immune system's defense against infections due to their strong antimicrobial properties and capacity for modulating the body's immunological responses. In this study, we successfully cloned and analyzed the yak DEFB123 gene sequence. Subsequently, we obtained recombinant protein DEFB123 (rDEFB123) through prokaryotic expression. Our results demonstrate that rDEFB123 effectively inhibits the growth of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Furthermore, rDEFB123 enhances the phagocytic activity of macrophages by regulating specific factors. In a mouse model infected with Klebsiella pneumoniae, the administration of rDEFB123 showed significantly lower levels of serum ALT and AST compared to the control group. Moreover, IFN-γ and IgG were significantly increased in the rDEFB123-treated groups, indicating an enhanced immune response. In the MAPKs signaling pathway of the infected mouse lungs, the expressions of JNK, TRAF2, TRAF6, MIF, and IL-1β genes were downregulated in the rDEFB123-treated groups. Moreover, the levels of p-JNK protein were significantly decreased in these groups as well. Klebsiella pneumoniae caused systemic infection with organ damage in mice. However, the administration of rDEFB123 suppressed the expressions of inflammatory factors, thereby mitigating organ injury and regulating the activity of apoptosis-related factors to enhance immunity. These findings provide valuable theoretical data for future exploration of the functionality and potential applications of DEFB123 in yak., 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.)

Lysozymes have gained attention for their antiseptic properties. In silico studies have shown that the enzyme containing lysM can act as an antibacterial agent. Binding of the lysM motif of rSELys to peptidoglycan and molecular dynamics simulations showed that the protein-ligand binding is very stable. rSELys (2016 bp) is a new recombinant glycoside hydrolase from the thermophilic bacterium Cohnella sp. A01 (PTCC number: 1921). Protein expression and purification, a single band with an apparent molecular weight of ∼74 kDa was observed by SDS-PAGE. The kinetic parameters were K m 1.163 mg/ml, Vmax 670.3 U/mg, k cat 1675.75 (S -1 ), and k cat /K m 1440.88 (M -1 S -1 ). Its optimum temperature was 55 °C and pH 8. Temperature stability also showed that the temperature of 50-60 °C retained more than half of its activity after 90 min. Based on the results, rSELys demonstrated antibacterial effects on both Gram-positive and Gram-negative strains, with inhibition zones of 11 and 9 mm, respectively. SEM analysis confirmed hydrolysis activity, the MIC was determined to be 31.25 μg/ml and 3.9 μg/ml, and MBC 0.97 μg/ml, respectively. CD and fluorescence studies showed that up to a temperature of 85 °C and a pH value of 8-12 no structural changes occur, and thermal stability protein was confirmed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements K m , V max , and k cat were determined to be 21 mM, 28 μM/min, and 39.6 S -1 , respectively. Thermal stability was assessed using a spectrofluorometer and showed T m value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC 50 values ranged from 28 to 45.5 μg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be -4.2 kcal mol -1 and - 4.4 kcal mol -1 , respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The serine protease gene was heterologously expressed in Escherichia coli BL21 (DE3) using the PET 28a vector. The purified enzyme was immobilized on a nanohybrid of amino graphene and chitosan. The characterization of synthesized nanohybrids and immobilized enzymes was confirmed by Fourier transform infrared (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and field emission scanning electron microscopy (FE-SEM). Immobilization increased the temperature optimum from 60 to 70 °C for both free and immobilized enzymes, while the optimal pH of the enzymes did not change post-immobilization (pH 8). The immobilized biocatalyst significantly enhanced thermal stability, as well as enzyme stability at significant pH ranges. After 30 days of storage, the immobilized enzymes exhibited approximately 83 % of their relative activity, while the free protease retained only 56 % of its initial activity. Stabilization also altered the kinetic parameters (increasing K m , decreasing Kcat/K m , and V max ) and thermodynamic parameters (increasing enzyme half-life and activation energy). The study's outcomes represent a significant advancement in the realm of enzyme synthesis and its stabilization using several combined technologies, including enzyme production with recombinant DNA technology based on gene synthesis, and its stabilization using a hybrid substrate synthesized from nanomaterials. Based on these findings, the immobilized recombinant enzyme has high potential for industrial use as an efficient and stable biocatalyst., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2024 Elsevier B.V. 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.)

We are investigating the glycolytic pathway in Pyrobaculum calidifontis whose genome sequence contains homologues of all the enzymes involved in this pathway. We have characterized most of them. An open reading frame, Pcal_0606, annotated as a putative phosphoglucose/phosphomannose isomerase has to be characterized yet. In silico analysis indicated the presence of more than one substrate binding pockets at the dimeric interface of Pcal_0606. The gene encoding Pcal_0606 was cloned and expressed in Escherichia coli. Recombinant Pcal_0606, produced in soluble form, exhibited highest enzyme activity at 90 °C and pH 8.5. Presence or absence of metal ions or EDTA did not significantly affect the enzyme activity. Under optimal conditions, Pcal_0606 displayed apparent K m values of 0.33, 0.34, and 0.29 mM against glucose 6-phosphate, mannose 6-phosphate and fructose 6-phosphate, respectively. In the same order, V max values against these substrates were 290, 235, and 240 μmol min -1  mg -1 , indicating that Pcal_0606 catalyzed the reversible isomerization of these substrates with nearly same catalytic efficiency. These results characterize Pcal_0606 a bifunctional phosphoglucose/phosphomannose isomerase, which displayed high thermostability with a half-life of ∼50 min at 100 °C. To the best of our knowledge, Pcal_0606 is the most active and thermostable bifunctional phosphoglucose/phosphomannose isomerase characterized to date., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pectinases are useful biocatalysts for pectic biomass processing and are extensively used in the food/feed, textile and papermaking industries. Two pectinase genes, a pectate lyase (SbPL1CE8) and a polygalacturonase (SbGH28GH105) were isolated from Segatella bryantii and functionally characterized. Recombinant rSbPL1CE8 was most active against polygalacturonic acid (PGA) and pectin with a 60 % degree of esterification, with k cat /K m values of 721.18 ± 64.77 and 327.02 ± 22.44 mL/s/mg, respectively. Truncated rSbPL1 acted as a mesophilic alkaline pectate lyase, which was highly resistant to inactivation by methanol and ethanol. The rSbPL1CE8 exclusively digested PGA and pectin into unsaturated digalacturonate (uG2), which was further converted into galacturonic acid by rSbGH28GH105. The rSbPL1CE8 was highly effective for saccharification of waste materials from Zea mays, Oryza sativa and Arachis hypogaea processing, and for ramie fiber degumming. This novel pectate lyase has great potential for application in industrial pectic biomass processing., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing-strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113-pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild-type strain AR113-pIB184., Results: The results demonstrated that the survival rate of AR113-pLLY01 was also significantly better than that of AR113-pIB184 after lyophilization. In addition, AR113-pLLY01 exhibited a significantly better protective effect than AR113-pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113-pLLY01 had a significant protective effect., Conclusion: This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T 50 , a 725% increase in half-life, a 5.5°C increase in T m , and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase., (© 2024 National Research Council Canada and The Authors. Biotechnology and Bioengineering by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH 2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca 2+ and Na + increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

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.)