Eukaryotic Expression Systems Research Articles

Improved protein splicing through viral passaging.

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEIntervening proteins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.

Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

A Review on cLF36, a Novel Recombinant Antimicrobial Peptide-Derived Camel Lactoferrin.

Lactoferrin is an antimicrobial peptide (AMP) playing a pivotal role in numerous biological processes. The primary antimicrobial efficacy of lactoferrin is associated with its N-terminal end, which contains various peptides, such as lactoferricin and lactoferrampin. In this context, our research team has developed a refined chimeric 42-mer peptide known as cLF36 over the past few years. This peptide encompasses the complete amino acid sequence of camel lactoferrampin and partial amino acid sequence of lactoferricin. The peptide's activity against human, avian, and plant bacterial pathogens has been assessed using different biological platforms, including prokaryotic (P170 and pET) and eukaryotic (HEK293) expression systems. The peptide positively influenced the growth performance and intestinal morphology of chickens challenged with pathogen bacteria. Computational methods and in vitro studies showed the peptide's antiviral effects against hepatitis C virus, influenza virus, and rotavirus. The chimeric peptide exhibited higher activity against certain tumor cell lines compared to normal cells, which may be attributed to the peptide's interaction with negatively charged glycosaminoglycans on the surface of tumor cells. Importantly, this peptide exhibited no toxicity against host cells and demonstrated remarkable thermal and protease stability in serum. In conclusion, while our investigations suggest that the chimeric peptide, cLF36, may offer potential as a candidate or complementary option to some available antibiotics, antiviral agents, and chemical pesticides, significant uncertainties remain regarding its cost-effectiveness, as well as its pharmacodynamic and pharmacokinetic characteristics, which require further elucidation.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

The crystal structure of human ferroptosis suppressive protein 1 in complex with flavin adenine dinucleotide and nicotinamide adenine nucleotide.

Ferroptosis is a recently discovered form of regulated cell death characterized by its distinct dependence on iron and the peroxidation of lipids within cellular membranes. Ferroptosis plays a crucial role in physiological and pathological situations and has attracted the attention of numerous scientists. Ferroptosis suppressive protein 1 (FSP1) is one of the main regulators that negatively regulates ferroptosis through the GPX4-independent FSP1-CoQ10-NAD(P)H axis and is a potential therapeutic target for ferroptosis-related diseases. However, the crystal structure of FSP1 has not been resolved, which hinders the development of therapeutic strategies targeting FSP1. To unravel this puzzle, we purified the human FSP1 (hFSP1) protein using the baculovirus eukaryotic cell expression system and solved its crystal structure at a resolution of 1.75Å. Furthermore, we evaluated the oxidoreductase activity of hFSP1 with NADH as the substrate and identified E156 as the key amino acid in maintaining hFSP1 activity. Interestingly, our results indicated that hFSP1 exists and functions in a monomeric state. Mutagenesis analysis revealed the critical role of the C-terminal domain in the binding of substrate. These findings significantly enhance our understanding of the functional mechanism of FSP1 and provide a precise model for further drug development.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Evaluation of the immunoprotective effects of porcine deltacoronavirus subunit vaccines

Porcine deltacoronavirus (PDCoV), a new porcine enteric coronavirus, has seriously endangered the pig breeding industry and caused great economic losses. However, a PDCoV vaccine is not commercially available. Therefore, new and efficient PDCoV vaccines must be developed without delay. In this study, we used the ExpiCHO eukaryotic expression system to express and purify the following 3 structural proteins of PDCoV: S, N and M. Subsequently, the level of humoral and cellular immunity induced by the S protein (immunization with the S protein alone) and a protein mixture (immunization with a mixture of S, N and M proteins) were evaluated in mice and piglets, respectively, and the performances of the 2 immunizations in a challenge protection test were assessed in piglets. The results showed that both the S protein and the protein mixture induced the production of high levels of specific IgG antibodies and neutralizing antibodies and effectively neutralized PDCoV-infected LLC-PK cells in vitro. Furthermore, compared with the S protein, the N and M proteins in the protein mixture promoted the expression of CD8+ T cells and IFN-γ, induced a stronger cellular immune response, and effectively protected 4/5 of the piglets from PDCoV infection. In conclusion, the results of this study showed that the N and M proteins play important roles in inducing an immunoprotective response. Using N and M antigens as effective antigenic components in the development of PDCoV vaccines in the future will effectively increase the immune efficacy of the vaccines.

Characterization of Prolyl-4-Hydroxylase Substrate Specificity Using Pichia pastoris as an Efficient Eukaryotic Expression System.

The use of eukaryotic expression systems facilitates the heterologous expression of complex eukaryotic proteins in their post-translationally modified and biologically active state, as a prerequisite for subsequent biochemical characterization and functional analysis. Here we describe the complete workflow for the expression of Arabidopsis thaliana prolyl-4-hydroxylases (P4Hs) in the methylotrophic yeast Pichia pastoris (renamed as Komagataella phaffii), for the extraction of the recombinant enzymes, purification by affinity chromatography, and characterization of P4H activity and specificity toward oligopeptide substrates by mass spectrometry. We expressed eight of the 13 Arabidopsis P4Hs and show that they are all active against proline-rich extensin-derived peptides. However, three of them differed in substrate specificity and were also able to hydroxylate the CLEL9 signaling peptide, featuring a single proline within its mature peptide sequence.

Expression and Anti-breast Cancer Effect of Immunotoxin scFv-Mmut

In this experiment, the Pichia Pastoris expression plasmid pPIC9K/scFv-Mmut was constructed, and the immunotoxin scFv-Mmut was successfully expressed in the eukaryotic expression system of Pichia Pastoris. The effect and mechanism of immunotoxin scFv-Mmut on inhibiting activity and inducing apoptosis of breast cancer cells were analyzed by in vitro assay. Through this experiment, it can be found that after the treatment of BT474 cells with a certain concentration of scFv-Mmut, the cell cycle will be affected and be blocked in the S phase, which proves that scFv-Mmut can induce the apoptosis of BT474 cells and has a significant anti-breast cancer effect.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Biochemical and Structural Characterization of a Uronic Acid Oxidase from Citrus sinensis

AbstractAldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. For this, effective biocatalysts are in demand. This work reports on the discovery, biochemical and structural characterization of a VAO‐type flavin‐containing carbohydrate oxidase from Citrus sinensis: URAOCs3. URAOCs3 could be overexpressed using prokaryotic and eukaryotic expression systems. Extensive biochemical characterization revealed that the enzyme displays a high thermostability and an exquisite selectivity for uronic acids, galacturonic acid and glucuronic acid. The enzyme was further investigated by determining the crystal structure. The selective oxidation of D‐galacturonic acid in a complex mixture was demonstrated, showing how URAOCs3 was found to be highly effective in selectively producing galactaric acid while leaving other carbohydrates untouched. In addition to the specific discovery of URAOCs3, these findings suggest that plant proteomes can be an interesting source for new biocatalysts.

A collagen-binding SIRPαFc fusion protein for targeted cancer immunotherapy

Collagen is abundant but exposed in tumor due to the abnormal tumor blood vessels, thus is considered as a tumor-specific target. The A3 domain of von Willebrand factor (vWF A3) is a kind of collagen-binding domain (CBD) which could bind collagen specifically. Previously we reported a chemosynthetic CBD-SIRPαFc conjugate, which could block CD47 and derived tumor-targeting ability by CBD. CBD-SIRPαFc conjugate represented improved anti-tumor efficacy with increased MHC II+ M1 macrophages, but the uncertain coupling ratio remained a problem. Herein, we produced a vWF A3-SIRPαFc fusion protein through eukaryotic expression system. It was examined at both molecular and cellular levels with its collagen affinity, uninfluenced original affinity to targets and phagocytosis-promoting function compared to unmodified SIRPαFc. Living imaging showed that vWF A3-SIRPαFc fusion protein derived the improved accumulation and retention in tumor than SIRPαFc. In the MC38 allograft model, vWF A3-SIRPαFc demonstrated a superior tumor-suppressing effect, characterized by increased MHC II+ M1 macrophages and T cells (particularly CD4+ T cells). These results revealed that vWF A3-SIRPαFc fusion protein derived tumor-targeting ability, leading to improved anti-tumor immunotherapeutic efficacy compared to SIRPαFc. Altogether, vWF A3 improved the anti-tumor efficacy and immune-activating function of SIRPαFc, supporting targeting tumor collagen as a possible targeted strategy.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Salmonella-Delivered COBRA-HA1 Antigen Derived from H1N1 Hemagglutinin Sequences Elicits Broad-Spectrum Protection Against Influenza A Subtypes

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4+ and CD8+ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4+ and CD8+ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.

Mechano-biomimetic hydrogel 3D cell cultivation as a strategy to improve mammalian cell protein expression.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Among which mammalian cell protein expression approach is the most powerful one, which can express complex proteins or genetic engineered biological drugs, such as PD-1. However, the high expense, which partially derives from its low protein yielding efficiency, limited the further application of such approach in large scale production of target proteins. To address this issue, we proposed a novel technique to promote the protein production efficiency of mammal cells without using conventional genetic engineered approaches. By placing 293T cells in a hydrogel 3D cell culture platform and adjusting the stress relaxation of the matrix hydrogel, cells formed multicellular spheroids by self-organization. In particular, the multicellular spheroids have a significantly enhanced ability to transiently express multiple proteins (SHH-N, PD-1 and PDL-1). We also examined in detail the mechanism underlying this phenomenon, and found that the reorganization of cytoskeleton during spheroids formation enhances the translation process of protein by recruiting ribosomes. Overall, this finding provides a novel approach for subsequent improvement of large-scale mammalian protein expression cell systems.

High expression of antimicrobial peptides cathelicidin-BF in Pichia pastoris and verification of its activity.

Antibacterial peptides are endogenous polypeptides produced by multicellular organisms to protect the host against pathogenic microbes, they show broad spectrum antimicrobial activities against various microorganisms and possess low propensity for developing resistance. The purpose of this study is to develop recombinant antibacterial peptide cathelicidin-BF by genetic engineering and protein engineering technology, and study its antibacterial activity in vitro and in vivo, so as to provide reference for the production and application of recombinant antibacterial peptide cathelicidin-BF. In this study, on account of Pichia pastoris eukaryotic expression system, we expressed and prepared antibacterial peptide cathelicidin-BF. Then, the minimum inhibitory concentration of antibacterial peptide cathelicidin-BF and the comparison with the antibacterial activity of antibiotics were determined through the antibacterial experiment in vitro. Chickens as infection model were used to verify the antibacterial peptide activity in vivo. The results show that the bacteriostatic ability of antibacterial peptide cathelicidin-BF is similar to that of antibiotics in certain concentration, and can reach the treatment level of antibiotics. Although the mode of administration of antibacterial peptide is still limited, this study can provide reference for the future research of antibacterial peptide.

Development of an Effective Neutralizing Antibody Assay for SARS-CoV-2 Diagnosis.

Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of Spodoptera frugiperda clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 μg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.

High-level production of Aspergillus niger prolyl endopeptidase from agricultural residue and its application in beer brewing

BackgroundProlyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost.ResultsHere, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer.ConclusionOur research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.