Recombinant Protein Expression Research Articles

Optimization of the intron sequences combined with the CMV promoter increases recombinant protein expression in CHO cells

To meet the requirements of the biopharmaceutical industry, improving the yield of recombination therapeutic protein (RTP) from Chinese hamster ovary (CHO) cells is necessary. The human cytomegalovirus (CMV) promoter is widely used for RTP expression in CHO cells. To further improve RTP production, we truncated the human CMV intron and further evaluated the effect of four synthetic introns, including ctEF-1α first, EF-1α first, chimeric, and β-globin introns combined with the CMV promoter on recombinant expression levels in transient and stably recombinant CHO cells. The results showed that the truncated, EF-1α first, chimeric, and β-globin introns can significantly improve stable transgene expression in CHO cells. The qPCR results indicated that the mRNA level of transgene increased through optimizing intron sequences combined with the CMV promoter. Transcriptomics analysis was performed and found that differential expression of genes involved in mRNA processing, RNA export from nucleus, cytoplasmic translation, transcriptional activation and cell cycle regulation. In conclusion, optimization of the intron sequences combined with the CMV promoter can achieve a higher yield of recombinant proteins in CHO cells. This will be valuable for generating CHO cell lines with high productivity for industrial applications.

Mining, characterization, and expression of a fructan sucrase for efficient conversion of soybean oligosaccharides

The high content of sucrose and raffinose reduces the prebiotic value of soybean oligosaccharides. Fructan sucrases can catalyze the conversion of sucrose and raffinose to high-value products such as fructooligosaccharides and melibiose. To obtain a fructan sucrase that can efficiently convert soybean oligosaccharides, we first mined the fructan sucrase gene from microorganisms in the coastal areas of Xisha Islands and Bohai Bay and then characterized the enzymatic and catalytic properties of the enzyme. Finally, recombinant extracellular expression of this gene was carried out in Bacillus subtilis. The results showed that a novel fructan sucrase, BhLS 39, was mined from Bacillus halotolerans. With sucrose and raffinose as substrates, BhLS 39 showed the optimal temperatures of 50 ℃ and 55 ℃, optimal pH 5.5 for both, and Kcat/Km ratio of 3.4 and 6.6 L/(mmol·s), respectively. When 400 g/L raffinose was used as the substrate, the melibiose conversion rate was 84.6% after 30 min treatment with 5 U BhLS 39. Furthermore, BhLS 39 catalyzed the conversion of sucrose to produce levan-type-fructooligosaccharide and levan. Then, the recombinant extracellular expression of BhLS 39 in B. subtilis was achieved. The co-expression of the intracellular chaperone DnaK and the extracellular chaperone PrsA increased the extracellular activity of the recombinant BhLS 39 by 5.2 folds to 17 U/mL compared with that of the control strain. BhLS 39 obtained in this study is conducive to improving the quality and economic benefits of soybean oligosaccharides. At the same time, the strategy used here to enhance the extracellular expression of BhLS 39 will also promote the efficient recombinant expression of other proteins in B. subtilis.

Development of an attenuated glutamine synthetase (GS) selection system for the stable expression of tissue plasminogen activator in CHO-K1 cells

Chinese hamster ovary (CHO) cells represent the most common host system for the expression of high-quality recombinant proteins. The development of stable CHO cell lines used in industrial recombinant protein production often relies on dihydrofolate reductase (DHFR) and glutamine synthetase (GS) amplification systems. Conventional approaches to develop stable cell lines lead to heterogeneous cell populations. Consequently, it is desirable to adopt innovative strategies to increase the efficiency of clone selection to reduce the time and effort invested in the cell line development process. Attenuating the selection marker gene is an effective strategy for isolating high-producing cells. In this study, we evaluated the efficiency of an attenuated glutamine synthetase selection system for the expression of human tissue plasminogen activator (t-PA) in CHO cells. We introduced an AU-rich element (ARE) at the 3’UTR of the glutamine synthetase coding sequence and employed a weak promoter (mSV40) for the expression of this gene. Subsequently, we analyzed the effect of ARE on the GS RNA levels, and recombinant t-PA expression. Our results demonstrate that the use of ARE significantly enhances the detection of high expressing cells compared to the control. Additionally, the t-PA expression level in GS-ARE clones was approximately 900-fold greater than those without the ARE.

Investigating the metabolic load of monoclonal antibody production conveyed to an inducible CHO cell line using a transfer-rate online monitoring system.

Shake flasks are a foundational tool in early process development by allowing high throughput exploration of the design space. However, lack of online data at this scale can hamper rapid decision making. Oxygen transfer rate (OTR) monitoring has been readily applied as an online process characterization tool at the benchtop bioreactor scale. Recent advances in modern sensing technology have allowed OTR monitoring to be available at the shake flask level. It is now possible to multiplex time-of-action (e.g., Induction, temperature shift, pH shift, feeding initiation, point of harvest) characterization studies by relying on careful analysis of OTR profile kinetics. As a result, there is potential to save time and capital expenditures while exploring process intensification studies though accurate and physiologically relevant online data. In this article, we detail the application of OTR monitoring to characterize the impact that recombinant protein production has on an inducible CHO cell line expressing Palivizumab. We then test out time-of-action studies to intensify protein production outcomes. We observe that recombinant protein expression causes a metabolic load that diminishes potential biomass growth. As a result, when compared to a control standard process, delaying induction and temperature shift has the potential to improve viable cell densities (VCD) by 2-fold thus increasing recombinant protein yield by over 30 %. The study also demonstrates that OTR can serve as a useful tool to detect cessation of exponential growth. Consequently, time-of-action points that are characteristic of inducible systems can be formulated accurately and reliably to maximize production performance.

Co-expression of the RPS6KB1 and PDPK1 genes for production of activated p70S6K1 using bac-to-bac baculovirus expression system

BackgroundRibosomal protein S6 kinase 1 (p70S6K1) is a member of the AGC family of serine/threonine kinases which plays a role in various cellular processes, including protein synthesis, cell growth, and survival. Dysregulation of p70S6K1, characterized by its overexpression and/or hyperactivation, has been implicated in numerous human pathologies, particularly in several types of cancer. Therefore, generating active, recombinant p70S6K1 is critical for investigating its role in cancer biology and for developing novel diagnostic or therapeutic approaches.MethodsThe baculovirus dual expression system was utilized, enabling the co-expression of two recombinant proteins in infected cells: (a) His-tagged S6K1 with a deletion of the C-terminal autoinhibitory motif and a phosphomimetic mutation at the mTORC1 phosphorylation site (T389D), and (b) untagged PDPK1 lacking the PH domain. The high activity of the purified kinase was confirmed by immunoblotting, as well as by Kinase-Glo and AlphaScreen kinase assays.ResultsEfficient expression of both recombinant proteins was achieved, resulting in highly pure preparations of His-tagged p70S6K1. The high activity of the purified kinase was confirmed through multiple kinase assays, demonstrating significantly higher levels of substrate phosphorylation compared to the tested commercial product.ConclusionHere, we report a reliable and efficient methodology for the expression and purification of highly active p70S6K1 (His-actS6K1) in quantity and quality that is suitable for biochemical/biophysical studies and high-throughput enzymatic assays. Our developed methodology offers a rapid and cost-effective approach for producing constitutively active His-actS6K1, which can be utilized in academic research and biotechnology.

A suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression.

Expression and purification of recombinant proteins in E. coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C-terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a completely scarless vector series with 9 different N-terminal tags. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.

Incorporation of recombinant proteins into extracellular vesicles by Lactococcus cremoris

Extracellular vesicles (EVs) are nanosized lipid bilayer particles released by various cellular organisms that carry an array of bioactive molecules. EVs have diagnostic potential, as they play a role in intercellular interspecies communication, and could be applied in drug delivery. In contrast to mammalian cell-derived EVs, the study of EVs from bacteria, particularly Gram-positive bacteria, received less research attention. This study aimed to investigate the production of EVs by lactic acid bacterium Lactococcus cremoris NZ9000 and to examine the impact of recombinant protein expression on their formation and protein content. Four different recombinant proteins were expressed in L. cremoris NZ9000, in different forms of expression and combinations, and the produced EVs were isolated using the standard ultracentrifugation method. The presence of vesicular structures (50–200 nm) in the samples was confirmed by transmission electron microscopy and by flow cytometry using membrane-specific stain. Mass spectrometry analyses confirmed the presence of recombinant proteins in the EVs fraction, with amounts ranging from 13.17 to 100%, highlighting their significant incorporation into the vesicles, together with intrinsic L. cremoris NZ9000 proteins that were either more abundant in the cytoplasm (ribosomal proteins, metabolic enzymes) or present in the membrane. The presence of the most abundant lactococcal proteins in EVs fraction suggests that protein cargo-loading of EVs in L. cremoris NZ9000 is not regulated. However, our data suggests that L. cremoris NZ9000 genetically engineered to express recombinant proteins can produce EVs containing these proteins in scalable manner. As L. cremoris NZ9000 is considered safe bacterium, EVs from L. cremoris NZ9000 could have several advantages over EVs from other bacteria, implying possible biotechnological applications, e.g. in therapeutic protein delivery.

Recombinant Protein Production and Purification of Rift Valley Fever Virus Nucleoprotein from Escherichia coli Expression Systems.

The recombinant expression and purification of viral proteins are a key component in the study of the immune response of viruses, as well as the creation of diagnostic techniques for the detection of viruses. For structurally simple proteins, one commonly used technique is the production of recombinant proteins in bacterial expression systems, which enable the large-scale synthesis and purification of recombinant viral proteins. In this technique, the cDNA encoding for a viral protein is cloned into a bacterial expression vector (with an appropriate purification tag), produced in a modified bacterial culture, and optimized for maximum protein production in a minimal amount of time. In this chapter, a protocol for the production of Rift Valley fever virus nucleoprotein is described. This protein has been previously shown to highly antigenic (and thus, is used in diagnostic tests), and has also been shown to be a potent inducer of the T-cell response following Rift Valley fever virus infection. The protocol outlined in this chapter describes the cloning of the cDNA of RVFV nucleoprotein into a bacterial expression vector, which also contains a fusion protein for optimal protein expression and solubilization, as well as a poly-histidine tag for efficient purification This chapter also describes the steps required for bacterial transformation, culture, lysis, purification and dialysis of RVFV nucleoprotein, resulting in a recombinant protein preparation, which can be upscaled to produce milligram quantities of protein product, which in turn can be used for downstream immunological and diagnostic applications.

Correlation between TCF7 and bladder cancer and feasibility of Erlotinib targeting in bladder cancer: Molecular mechanism and expression of TCF7 recombinant protein.

The primary objective of this study was to conduct a comprehensive analysis of the mechanism by which TCF7 recombinant protein operates, as well as to examine its expression patterns within bladder cancer cells. This research seeks to establish a new theoretical framework and provide experimental data that could advance the field of molecular targeted therapy for bladder cancer. Erlotinib, a well-known targeted therapy drug, was administered to the bladder cancer cells, and we evaluated its antitumor effects through various assays such as cell proliferation, apoptosis, and cell cycle analysis. To delve deeper into the mechanisms of action associated with the TCF7 recombinant protein, we conducted extensive analyses of signaling pathways as well as gene expression profiles. The findings indicated a significant correlation between the expression levels of TCF7 in bladder cancer cells and the tumor's malignancy grade. Following the treatment with Erlotinib, we observed a marked inhibition of cell proliferation, an increase in apoptotic activity, and notable alterations in cell cycle distribution. The results suggested that the molecules of the TCF7 recombinant protein could potentially influence the biological characteristics and behavior of bladder cancer cells by modulating specific signaling pathways, particularly the Wnt/β-catenin signaling pathway. Further analysis of the gene expression profiles also enabled us to identify downstream target genes associated with TCF7, ultimately shedding light on its specific mechanisms of action in the context of bladder cancer.

Bunyaviruses: Expression of Recombinant Glycoproteins from Insect Cells.

High-density suspension cultures of insect cells offer a scalable and serum-free system for the expression of recombinant proteins. Rift Valley fever virus (RVFV), an arthropod-borne virus spread by mosquitoes, contains two envelop glycoproteins Gn and Gc. These glycoproteins are crucial for eliciting neutralizing antibodies that can offer protection against RVFV infection. Here, we describe the production of individual RVFV glycoproteins and empty RVFV particles using S2 Drosophila insect cells, followed by purification and analysis of the purified proteins.

A mutant complement factor H (W1183R) enhances proteolytic cleavage of von Willebrand factor by ADAMTS-13 under shear.

A loss-of-functional mutation (W1183R) in human complement factor H (CFH) is associated with complement-associated hemolytic uremic syndrome; mice carrying a similar mutation (W1206R) in CFH also develop thrombotic microangiopathy but its plasma von Willebrand factor (VWF) multimer sizes were dramatically reduced. The mechanism underlying such a dramatic change in plasma VWF multimer distribution in these mice is not fully understood. To determine the VWF and CFH interaction and how CFH proteins affect VWF multimer distribution. We employed recombinant protein expression, purification, and various biochemical and biophysical tools. Purified recombinant W1183R-CFH but not wild-type (WT) CFH protein enhanced the proteolytic cleavage of both peptidyl and multimeric VWF substrates by recombinant ADAMTS-13 in a concentration-dependent manner. Microscale thermophoresis assay demonstrated that both W1183R-CFH and WT-CFH proteins bound various VWF fragments (eg, AIM-A1, A1-A2-A3, D'D3, D'D3-A1, and D'D3-A1-A2) with high affinities. Optical tweezer experiments further showed a concentration-dependent alteration in the contour length (Lc) and the persistent length (Lp) following pulling VWF-A2 domain in the presence of W1183R-CFH or WT-CFH protein. AlphaFold experiments revealed conformational changes in the VWF-A2, particularly the central region where the cleavage bond resides following addition of W1183R-CFH or WT-CFH protein. These results demonstrate for the first time that W1183R-CFH but not WT-CFH protein enhances the proteolytic cleavage of VWF by ADAMTS-13 under shear. This may be achieved by mechanic-induced conformational changes of the central A2 domain, leading to an enhanced cleavage of Tyr1605-Met1606 bond by ADAMTS-13 under pathophysiological conditions.

Blocking virus infection with BacMam virus delivery bovine interferon-α gene

ABSTRACT Interferon-αs (IFN-αs) are crucial cytokines for inducing protective antiviral responses. The baculovirus-mediated gene transduction of mammalian cells (BacMam) is an efficient delivery tool for recombinant protein expression in mammalian cells. This study focuses on the delivery of bovine IFN-α (BoIFNα) using the BacMam system. A recombinant pACEMam1-BoIFNα bacmid was constructed, and a recombinant BacMam-BoIFNα virus was obtained. After transducing HEK293T cells with this virus, BoIFNα protein was successfully secreted into the cell supernatant, displaying antiviral activities against VSV, BPIV3, BEV, and BVDV in bovine-derived cells. Additionally, the BacMam-BoIFNα virus inhibited the replication of these viruses in MDBK cells, induced the transcription of ISGs, and the expression of M × 1 in both MDBK and BT cells. These induction effects were significantly reduced following treatment with a JAK1 inhibitor, suggesting that the BacMam-BoIFNα virus exerts antiviral activity in MDBK cells through JAK-STAT signaling pathway. Furthermore, the study found that the BacMam-BoIFNα virus significantly inhibited the replication of BPIV3 in the lung and trachea of mice. Overall, this study demonstrates that the BacMam-BoIFNα virus have antivirus activities both in vitro and in vivo, signaling through JAK-STAT pathway, and provides an efficient interferon gene delivery tool for combating bovine viral infections.

Development of high-performance inducible and secretory expression vector and host system for enhanced recombinant protein production

The production of lipopolysaccharide (LPS)-free recombinant proteins from culture supernatants is of great interest to biomedical research and industry. Due to the LPS-free cell wall structure and the well-defined secretion factor B (SecB)-dependent secretion pathway, Gram-positive bacteria are a superior alternative to Escherichia coli expression systems. However, the lack of inducible expression systems for high yields has been a bottleneck. To address this, we developed the pKS81 plasmid, featuring the uhpT (glucose-6-phosphate [G6P] transporter) promoter for high expression of recombinant proteins induced by extracellular G6P via a three-component hexose phosphate transport regulatory system (HptARS), the N-terminal SecB-dependent signal peptide sequence for recombinant protein secretion, and the C-terminal 8 × histidine tag for purification by nickel affinity chromatography. We also generated an expression host strain, Staphylococcus aureus LAC9, lacks the uhpT gene and harmful superantigen and leukotoxin genes, allowing for constitutive HptARS activation by extracellular G6P and increased safety, respectively. Using the pKS81 plasmid, we successfully achieved high yields of prokaryotic (staphylococcal leukotoxin E) and eukaryotic (human annexin A2 protein tagged with mouse IgG1) recombinant proteins, up to 900 mg/L. Our newly established inducible and secretory expression system provides for efficient production and easy purification of LPS-free recombinant proteins, making it valuable for biomedical research and industrial applications.

Constitutive Overexpression of CRZ1 in Trichoderma reesei Reveals Its Ability to Enhance Recombinant Lipase Production.

The filamentous fungus Trichoderma reesei is extensively utilized in the realm of recombinant protein expression owing to its well-established protein production systems. However, the potential for efficient and convenient protein production in T. reesei has not been fully harnessed. To further increase the production of recombinant lipase Candida antarctica lipase B (CalB), we overexpressed seven transcription activators and found that overexpression of the calcineurin CRZ1 could significantly enhance CalB production by 2.08-fold, and then through employing a synthetic strong promoter Pcbh7IR and knocking out ace1, a competitor of crz1, CalB production was further increased 4.2-fold. Furthermore, we found that increasing the Ca2+ concentration enabled the enhancement of CalB production in the CRZ1-overexpressing strains. Additionally, transcription analysis revealed a significant upregulation of calcium-related pathway genes and ER chaperone genes in T. reesei, revealing that overexpression of CRZ1 can increase the production of the recombinant protein by activation of the calcium signaling pathway. In this study, by systematically manipulating the recombinant protein expression module and its regulatory transcription activators, we found a positive effect of crz1-mediated calcium channels in boosting the expression of the recombinant protein.

Oxidative metabolism mechanism of terpenoid compound ZQ-8 by cytochrome P450 enzyme in Helicoverpa armigera.

In our previous research, we identified that treatment of Helicoverpa armigera with ZQ-8 led to upregulation of CYP450 genes. To clarify the metabolic pathway of ZQ-8, this study analyzed the expression of CYP450 genes and proteins in H. armigera after ZQ-8 treatment through transcriptomics and proteomics. Molecular docking, recombinant protein expression, and surface plasmon resonance techniques were employed to investigate the interactions between ZQ-8 and P450 proteins. The oxidative reduction related pathways were significantly enriched in H. armigera larvae treated with ZQ-8, with an increase in the expression of CYP6B2 and CYP6B6 genes. The CYP6B2 and CYP6B6 proteins exhibited significant expression following ZQ-8 treatment. ZQ-8 demonstrated rapid binding and stable dissociation characteristics with CYP6B6, characterized by a dissociation constant (KD) of 88.15 μM. In contrast, ZQ-8 also showed rapid binding and dissociation with CYP6B2, but with a lower KD of 74.77 μM indicating that CYP6B2 has a stronger binding affinity for ZQ-8 compared to CYP6B6, and is capable of oxidizing ZQ-8 to the corresponding carboxylic acid. This study provides a reference for the metabolism and mechanism of action of ZQ-8 as a potential drug molecule, laying the foundation for future drug design and optimization, paving the way for environmentally sustainable pest control strategies and reducing reliance on traditional chemical pesticides.

Optimized high-yield expression of envelope glycoprotein domain III from dengue virus serotypes 1 to 4.

Dengue virus (DENV) envelope glycoprotein Domain III (EDIII) is critical for viral entry, highly immunogenic, and induces robust neutralizing antibody response. It is a prominent candidate for designing subunit-based vaccines and can also be harnessed as an antigenic bait for isolation of neutralizing human mAbs. Here, we describe an optimized method for high-yield expression of recombinant domain EDIII protein from DENV serotypes 1 to 4 in different Escherichia coli (E. coli) expression strains. The DENV EDIII proteins show differential expression patterns in tested E. coli expression strains. The structural integrity of the purified and refolded proteins is further validated using the Circular Dichroism (CD) spectroscopy and Fourier Transform Infrared (FTIR) spectroscopic analysis. The functional validation of the purified refolded DENV EDIII proteins through Enzyme-linked Immunosorbent Assay (ELISA) and co-immunoprecipitation (Co-IP) exhibits efficient binding with a well-characterized humanized neutralizing mAb 513. Further, we compared the potency of purified EDIII in blocking viral through competitive inhibition assay. Our study highlights that a universal expression system may not be an ideal approach for all DENV EDIII protein expression.

Matrix attachment regions enhance transgene expression by manipulating position-dependent effects in stably transfected CHO-K1 cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluorescent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recombinant protein production in CHO-K1 cells. This insight underscores the importance of strategic MAR incorporation in vector design for optimized recombinant protein expression.

Cloning of the GOX-Xho Gene IPBCC 08.610 into Plasmid pTA2 and Its Characterization

Glucose oxidase (GOX) from Aspergillus niger catalyzes the oxidation of β-D-glucose to δ-gluconolactone and hydrogen peroxide, making it valuable for industrial applications. Intracellular GOX exhibits higher activity than its extracellular counterpart. This study focuses on enhancing the extracellular production of GOX through recombinant DNA technology. This study aimed to reconstruct the GOX gene by adding XhoI sites at both ends, inserting a glu-ala-glu-ala spacer at the 5' end, and introducing an XbaI site at the 3' end. These modifications facilitate the cloning of the GOX-Xho gene into the pTA2 vector and its subsequent ligation into the pPICZαB expression vector, allowing for extracellular production of GOX through fusion with the α-mating factor (α-MF) signal peptide. The GOX-Xho gene was successfully amplified, cloned, and characterized. The pTA2-GOX-Xho recombinant plasmid was verified through sequencing and restriction analysis, confirming the present and correct orientation of the 1797 bp GOX-Xho gene. However, sequencing revealed several point mutations, necessitating further computational analysis to predict their impact on the enzyme's structure and function before recombinant protein expression.

Characterization of a Salmonella abortus equi phage 4FS1 and its depolymerase.

The significant economic losses caused by S. abortus equi in donkey husbandry have increased interest in exploring the potential of phages and their enzymes as control strategies. In this study, a S. abortus equi phage, designated 4FS1, was isolated from sewage at a donkey farm. Transmission electron microscopy (TEM) revealed a typical icosahedral head and a long, non-contractile tail. It exhibited a short latent period of 20 min and a burst size of 160 plaque-forming units (PFU) per cell. It demonstrated a broad host range, infecting 36 out of 60 salmonella strains, with an optimal multiplicity of infection (MOI) of 0.01 for S. abortus equi S1. The phage titer remained stable at 109 PFU/mL between 37°C and 50°C and exceeded 108 PFU/mL at pH from 5.0 to 10.0. After 1 h of UV exposure, the titer remained at 107 PFU/mL and showed no significant variation across NaCl concentrations from 2.5 to 15%. The genome of phage 4FS1 consists of a 42,485 bp linear double-stranded DNA molecule with a G + C content of 49.07%. Of the 56 predicted open reading frames (ORFs), 32 were functional annotated, with no virulence or drug resistance genes identified. ORF36 was predicted to encode a depolymerase responsible for endorhamnosidase activity. Recombinant expression of the Dpo36 protein in prokaryotes significantly reduced biofilm formation and removal. Combined with healthy donkey serum, Dpo36 inhibited bacterial growth in vitro and enhanced the survival rates of mice infected with S. abortus equi. These findings highlight the promising potential of phages and their depolymerases as novel therapeutic agents against S. abortus equi.

Removal, detection, and limits of endotoxin in the industry of recombinant proteins

With the advancement of synthetic biology, recombinant proteins are poised to play a significant role in medical applications. The scaled manufacturing is a pillar for the extensive application and development of recombinant proteins across various fields. In the large-scale production process of recombinant proteins, the removal and detection of endotoxins are essential to reduce their levels to safe thresholds in the final products. Currently, establishing stringent endotoxin limits for different recombinant protein products is a crucial aspect of safety assessment. This review begins by shedding light on the pathogenicity of endotoxins and discusses the methods for the removal and determination of endotoxins during the production processes of recombinant proteins. Subsequently, this review summarizes the endotoxin limits in industries such as biologics, medical devices, and human recombinant DNA products, particularly those in recombinant protein injection products. It is highlighted that regardless of whether the hosts for recombinant protein expression are bacteria or not, endotoxin testing is required for the final products of injectable recombinant proteins, and compliance with relevant industry standards is necessary. This review aims to provide a reference for the research on endotoxins in the large-scale production process of recombinant proteins.