Expression System Research Articles

Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.

Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during Escherichia coli cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.

Rapid Screening of Plastic-Degrading Enzymes Using an Optimized Cell-Free Protein Synthesis Platform.

The accumulation of plastic waste poses a significant environmental challenge, necessitating the development of efficient plastic-degrading enzymes for bioremediation and recycling. However, traditional enzyme engineering approaches rely on microbial expression systems and are time-consuming and prone to unintended interactions between host cells and recombinant circuits. To address these limitations, a cell-free protein synthesis (CFPS) platform was developed for rapidly screening plastic-degrading enzymes. Using CFPS, cutinase and PET-degrading enzymes (PETases) were successfully synthesized, and their catalytic activities were assessed using polymer-containing agar plates. Clear degradation halos were observed for cutinase and PETase on polycaprolactone (PCL)-containing and bis (2-hydroxyethyl) terephthalate (BHET)-containing plates, respectively. The optimization of CFPS conditions revealed that enzyme synthesis efficacy was higher at room temperature than at 37°C. The screening of PETase variants (C3 N1377, Mipa-P, and C5 N1251), synthesized using the CFPS platform, demonstrated that the catalytic activity of Mipa-P was the highest and surpassed that of IsPETase. This finding was further validated through purified enzyme analysis. Our results establish CFPS as a rapid, scalable, and cell-free alternative platform for screening and optimizing plastic-degrading enzymes, facilitating advancements in enzymatic plastic recycling.

A transposon-based transient transfection system in CHO-K1 cells enables quality prediction of stable cell line proteins.

In biologics drug discovery, transient protein expression is widely used to rapidly produce biologics, thereby accelerating the identification of lead candidates. However, the accuracy and consistency of predictingfurther product quality in large-scale production needs to be considered, especially with respect to physicochemical properties and posttranslational modifications. With this in mind, a transient expression system utilizing Chinese hamster ovary K1 (CHO-K1) has been established, whichintegrates high expression capability with quality profiles similar to those of the protein produced by stable cell lines. A well-designed vector containing transposon elements overcomes the blindness of random integration andensuresthe sustained viabilityof cells and production capability, thus addressing the critical bottlenecks in classical transiently transfected workflows. Combined with the optimization of various transfectionparameters, the customized platform achieved a titer over 1.5g/L in the production of abispecific antibodywhile maintaining a proportion of fragments, aggregates and glycosylation patterns that are comparable to those of the stable cell line protein.More importantly, this platform also demonstrated reliability in terms of quality across diverse antibody formats. This innovative protein expression platform bridges the gap between transient and stable expression on the basis of CHO-K1, ensuring the consistency of host cell types throughout the antibody discovery and development process.

Phage Cell Wall Binding Domain for Gram-Negative Bacteria and Its Application in Detection of Acinetobacter baumannii.

The cell wall binding domain (CBD) of endolysin plays an irreplaceable role in the propagation cycle of phages as it can specifically bind with the cell wall of target bacteria. Although it is a promising agent for recognizing bacteria, this domain is usually found in the endolysin of phages infecting Gram-positive bacteria, hindering its application for the detection of Gram-negative bacteria. Herein, we isolated a highly virulent phage termed as Abp18 for infecting Acinetobacter baumannii (A. baumannii), and found that its endolysin has a CBD termed as CBD18 for binding Gram-negative bacteria. This CBD was acquired using an Escherichia coli (E. coli) expression system, which can bind with 16 of the 18 tested A. baumannii strains with a recognition rate of approximate 89%. In accordance with the result of the turbidity reduction experiment, CBD18 has no lytic activity and thus can be used as a capture agent for A. baumannii. Furthermore, its fusion body with green fluorescent protein was acquired by fusion expression technology, which can be adopted as a high-performance signal probe for tracing A. baumannii. These two proteins were used to establish a sandwich method for detecting this pathogen with a linear range of 4.0 × 102 to 4.0 × 108 CFU/mL and a detection limit of 1.7 × 102 CFU/mL. The practicability of the CBD-based method was validated by detecting A. baumannii spiked in diverse types of real samples, with recoveries of 89-120%. The pioneering study paves an avenue for the usage of phage CBD in recognizing Gram-negative bacteria.

Purification and characterization of recombinant neuraminidase as a potentially broadly protective influenza virus vaccine candidate.

Purification and characterization of recombinant neuraminidase as a potentially broadly protective influenza virus vaccine candidate.

Production of functional human potassium channel protein KV1.5 in an Escherichia coli-based cell-free protein synthesis system.

KV1.5 is a member of the voltage-gated potassium ion channel family, which plays important roles in cell excitability, ion homeostasis and cell signaling. It is very difficult to rapidly obtain functional ion channel proteins in large quantities. Here, we report the utilisation of an Escherichia coli cell-free protein expression system to efficiently express KV1.5 in a soluble form over a 16 h period, achieving a yield of up to 1 mg·mL-1. The synthesised KV1.5 was purified using a HisTrap HP column to capture the 6 × his tags in the presence of detergent FC-14. The resulting KV1.5 exhibited α-helical secondary structures as assayed by circular dichroism spectroscopy, and demonstrated binding affinity for its agonist benzocaine through microscale thermophoresis measurements. Both, excitatory and inhibitory effects of benzocaine and vernakalant on reconstituted KV1.5-liposomes was measured using H+-dependent quenching of a fluorescent dye, 9-amino-6-chloro-2-methoxyacridine, to monitor the flux of K+. Overall, our findings demonstrated that the functional human ion channel KV1.5 could be heterologously synthesised via an E. coli-based cell-free protein expression system, which will expand the application scope of the cell-free protein expression system for structure and drug high-throughput screening within the entire voltage-gated potassium channel family.

Comparative analysis of convergent and divergent T7 RNA polymerase promoters for the synthesis of dsRNA in vivo and in vitro.

Double-stranded RNA plays a key role in various biological processes. The discovery of RNA interference, a gene-silencing mechanism, revolutionised the study of gene function. dsRNA has since been used in novel therapeutics and as an agricultural biocontrol alternative to chemical pesticides. Microbial production typically involves expression systems with convergent T7 promoters. However, convergent transcription from DNA-dependent RNA polymerases can lead to transcriptional interference. In this study, we designed multiple plasmid DNA constructs to investigate the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo in E. coli, prior to dsRNA yield quantification and analysis of product quality. We demonstrate that higher yields of larger dsRNA are typically obtained using convergent promoters during in vivo production. A typical fold increase of 2.1 was obtained for dsRNA > 400 bp. However, production of smaller dsRNA (< 250 bp) by divergent promoters resulted in increased yields (2.2 fold). Furthermore, our data demonstrates that in vitro transcription production of dsRNA using divergent T7 promoters results in significantly higher yields of dsRNA, with a maximum fold increase of 6.46. Finally, independent of size, we demonstrate that dsRNA synthesised from DNA templates with multiple transcriptional terminators, compared to run-off transcription, improved the quality and purity of dsRNA due to decreased formation of dsRNA multimers or aggregates. This study highlights alternative optimal strategies for the production of a wide range of different sized dsRNA both in vitro and in microbial systems.

Enhancing Mycobacterium tuberculosis-Ag85B immunogenicity by fusing with human Fcγ1 (Ag85B:hFcγ1).

Enhancing Mycobacterium tuberculosis-Ag85B immunogenicity by fusing with human Fcγ1 (Ag85B:hFcγ1).

Soluble expression and functional characterization of multi-subunit hemoglobins from the blood clam, Tegillarca granosa.

Soluble expression and functional characterization of multi-subunit hemoglobins from the blood clam, Tegillarca granosa.

Regioselective Hydration of Terpenes with Cofactor-Independent Carotenoid 1,2-Hydratase.

Terminally hydrated terpenes are highly sought-after compounds in the flavor and fragrance industries. However, their selective synthesis remains a considerable challenge in catalysis. Regioselective hydration of non-activated C-C double bonds is typically hindered by poor selectivity and low atom efficiency in conventional methods. In this study, we harness the underexplored potential of the acyclic carotenoid 1,2-hydratase fromRubrivivax gelatinosusIL144, employing it as a whole-cell biocatalyst for cofactor-independent terminal hydration of a diverse range of terpenes. This enzyme demonstrates exceptional activity across more than twenty C12-C20terpenes and shows notable tolerance to various functional groups, establishing it as a valuable tool for sustainable organic synthesis. We emphasize the critical influence of expression system choice in maximizing enzymatic performance, enabling high-yield transformations on the gram scale. Through a combination of homology modeling, consensus analysis, and targeted mutagenesis, essential residues involved in catalytic activity were identified. Notably, enhanced catalytic efficiency was only achievable through the epistatic effect of three specific mutations. These findings highlight the biocatalytic potential of acyclic carotenoid hydratase, offering a green and efficient route to the production of valuable tertiary alcohols.

Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain

BackgroundAspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.ResultsIn this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48–72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 − 1328 U/mL, ~ 1627. 43 − 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.ConclusionsOur results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.

Characterization of a Potential Therapeutic Anti-Canine PD-1 Single Domain Antibody Produced in Yeast

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development.

PET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli, validated by production of functional human ACE2.

The pET28g plasmid is a new tool for protein expression in Escherichia coli. It was derived from pET28a by replacing the traditional multiple cloning site with a Golden Gate cassette containing the lacZα reporter gene. The assembly of pET28g is based on Golden Gate cloning, and it was designed as an extension of the MoClo Toolkit (Addgene Kit #1000000044). The pET28g plasmid, along with the level 0 modules developed in this work, expands the existing pET plasmid collection and offers a versatile and modular system for protein expression. These tools facilitate the establishment of efficient pipelines for the preparation and testing of multiple constructs, enabling the optimization of conditions to achieve high levels of soluble protein expression. As proof of concept, we successfully produced and purified the peptidase domain of human ACE2 using the pET28g system. Notably, this represents the first report of the successful purification of a functional human ACE2 protein from the Escherichia coli soluble protein fraction. This lab protocol, which is complemented by the protocol at www.protocols.io (https://www.protocols.io/view/preparation-of-level-0-modules-for-golden-gate-ass-j8nlk9e75v5r/v1), provides a comprehensive guide to understanding the nomenclature of the pET28g system and includes step-by-step instructions for the preparation of level 0 modules and the assembly of the final plasmid to express constructs of interest using pET28g.

Construction of a cell model with membrane-surface expression of canine PD-1 and PD-L1 proteins in 293T cells by using eukaryotic expression systems

ABSTRACT Breakthrough progress has been made in the molecular mechanism research and clinical application of PD-1/PD-L1 in the regulation of immunosuppression and tolerance mainly in human and mouse fields, but is relatively slow in other species. The eukaryotic expression vectors pECFP-Fc-1 and pEYFP-Fc-L1 for high expression of canine PD-1 and PD-L1 proteins were constructed and transfected into human embryonic kidney 293 T cells. Fluorescence microscopy, laser scanning confocal microscopy, and immunofluorescence technology were used to identify the expression and membrane localization of the target proteins in human embryonic kidney 293 T cells. The binding activity of the target proteins expressed on the model cell was identified by eukaryotic expression vector co-transfection and immunocoprecipitation. The results showed that canine PD-1 and PD-L1 proteins were expressed on the membrane surfaces of their respective positively transfected cells. The cell membrane complex was further analyzed by co-immunoprecipitation technology, PD-L1 protein components were successfully detected in the pull-down complex of canine PD-1 antibody, and the two target proteins expressed in the model cells showed good mutual binding activity. Further research is needed to evaluate high throughput and a reliable method for screening drugs that block the PD-1 and PD-L1 pathway.

Regulation of the tagatose catabolic gene cluster and development of a tagatose-inducible expression system in the probiotic Escherichia coli Nissle 1917

BackgroundThe probiotic Escherichia coli Nissle 1917 (EcN) is a promising microbial chassis for therapeutic and industrial applications. However, its broad utility is limited by a lack of reliable inducible gene expression systems that precisely control gene expression.ResultsWe developed a tagatose-inducible expression system in EcN using D-tagatose, a naturally occurring sugar with established safety in humans, as a metabolizable inducer. Through differential RNA sequencing and sequence analysis, we identified the key regulatory elements governing D-tagatose catabolism in EcN and demonstrated that the DeoR family regulator (TagR) functions as a tagatose-responsive repressor. The developed system exhibited a strong dose-dependent response to D-tagatose, ensuring uniform and tunable gene activation across cell populations. Additionally, a catabolite repression-enabled auto-induction strategy facilitated robust biomass accumulation, followed by targeted protein production. This expression system was successfully applied to overexpress recombinant proteins under both aerobic and anaerobic conditions.ConclusionsD-Tagatose is a naturally occurring low-calorie sugar that can serve as an inducer in vivo, including within the human gut microbiome. Thus, the tagatose-inducible expression system provides EcN with an additional tunable option for gene regulation, which may be valuable in applications such as synthetic biology and metabolic engineering.

Hydrophobin-Fused SARS-CoV-2 RBD production in Nicotiana benthamiana L. for COVID-19 serology

IntroductionThe global health crisis underscores the need for accessible and cost-effective biomedical solutions, especially in resource-limited settings. Plant-based transient expression systems provide a viable method for producing recombinant proteins, such as the SARS-CoV-2 receptor-binding domain (RBD), for diagnostic applications. The addition of hydrophobin enhances protein purification efficiency and cost-effectiveness.MethodsThe study produced recombinant SARS-CoV-2 RBD fused to hydrophobin I (RBD-S-SCV2-HFBI) in Nicotiana benthamiana L. Immunogenicity was evaluated using serum from Swiss mice immunized with viral supernatant. Serum samples from COVID-19 patients were tested for immunogenicity of RBD-S-SCV2-HFBI using SDS-PAGE, Western blotting, and ELISA. Statistical analysis included descriptive statistics and one-way ANOVA with Kruskall-Wallis.ResultsThe RBD-S-SCV2-HFBI protein was successfully expressed in Nicotiana benthamiana L., demonstrating high immunogenicity. It was significantly recognized by IgG and IgM antibodies in serum samples from COVID-19 patients. The hydrophobin tag improved protein aggregation and purification, enabling efficient and scalable production.ConclusionThe study validates the feasibility of using RBD-S-SCV2-HFBI for diagnostic applications. Plant-based systems offer a rapid and cost-effective platform for producing high-quality recombinant proteins, providing scalable solutions to global health emergencies, particularly in resource-limited contexts.

Identification of a Novel Antibacterial Function of Mammalian Calreticulin

Calreticulin is a highly conserved and multifunctional molecular chaperone ubiquitously expressed in humans and animals. Beyond its well-established roles in calcium homeostasis, protein folding, and immune regulation, recent studies in aquatic species have suggested a previously unrecognized antimicrobial function of calreticulin. These findings raise the question of whether calreticulin also exerts antibacterial activity in terrestrial mammals, which has not been systematically investigated to date. To address this knowledge gap, we successfully constructed and expressed recombinant goat calreticulin using the Pichia pastoris expression system, yielding a protein of over 99% purity that predominantly exists in dimeric form. Functional assays demonstrated that both recombinant goat and human calreticulin exhibited preliminary inhibitory activity against Escherichia coli, Salmonella typhimurium, and Pasteurella multocida. Calreticulin was capable of binding to these three bacterial species as well as bacterial lipopolysaccharides (LPS). Notably, in the presence of Ca2+, calreticulin induced bacterial aggregation, indicating a potential mechanism for limiting bacterial dissemination and proliferation. Given the high anatomical, genetic, and physiological similarity between goats and humans—particularly in respiratory tract structure and mucosal immune function—neonatal goats were selected as a relevant model for evaluating the in vivo antimicrobial efficacy of calreticulin. Accordingly, we established an intranasal infection model using Pasteurella multocida to assess the protective role of calreticulin against respiratory bacterial challenge. Following infection, calreticulin expression was markedly upregulated in the nasal mucosa, trachea, and lung tissues. Moreover, intranasal administration of exogenous calreticulin significantly alleviated infection-induced pathological injury to the respiratory system and effectively decreased bacterial loads in infected tissues. Collectively, this study systematically elucidates the antimicrobial activity of calreticulin in a mammalian model and highlights its potential as a natural immune effector, providing novel insights for the development of host-targeted antimicrobial strategies.

Human endogenous retrovirus-K envelope protein is aberrantly expressed in serous ovarian cancer and promotes chemosensitivity via NF-κB/P-glycoprotein pathway inhibition

BackgroundApproximately 100,000 human endogenous retroviruses (HERV) are integrated into the human genome. Most HERVs are not expressed; however, transcription within the family HERV-K, a class II beta-retrovirus, occurs in specific cancers. Herein, we investigated HERV-K env protein expression and its clinical and molecular significance in serous ovarian cancer.MethodsProtein expression was assessed via immunohistochemistry and QuPath digital analysis in 24 normal, 10 benign, 13 borderline and 72 cancerous serous ovarian tissues. Clinicopathological parameters were obtained from medical records. Transcriptomes were evaluated using strand-specific reverse transcription-PCR and sequencing. Antiproliferative activities were explored using MTT and colony formation assays. A stable transfection expression system and siRNA gene silencing were used. Resistant cell lines were established using the paclitaxel concentration gradient method and chemoresponsiveness was evaluated by measuring the IC50.ResultsHERV-K env was absent in normal ovarian epithelia and benign tumors but was detected in 37.5% (27 of 72) of serous carcinomas and 61.5% (8 of 13) of borderline tumors. Unexpectedly, HERV-K env was observed in lymphocytes only in a subset of HERV-K env-positive tumors: 18 of 27 invasive and 1 of 8 borderline tumors. HERV-K env-positivity was significantly associated with chemosensitivity, although the prognosis was unaffected. HERV-K type I (K101, K102, and K103) and II (K108 and K115) were transcribed in cultured ovarian cancer cells and tissues but not in their paclitaxel-resistant derivatives. Forced expression of HERV-K env in paclitaxel-resistant cells suppressed cellular proliferation and resensitized cells to paclitaxel by inhibiting NF-κB/P-glycoprotein. siRNA-mediated HERV-K env knockdown restored paclitaxel-resistance by recovering NF-κB/P-glycoprotein.ConclusionHERV-K env was expressed in serous ovarian carcinoma and significantly associated with chemosensitivity. HERV-K env attenuated NF-κB/P-glycoprotein, a mechanism of chemoresistance. Hence, it could have therapeutic potential in chemoresistant ovarian cancers.

Extracellular Expression and Diagnostic Potential of Dengue Virus Type 3 E Protein Domain III Through Codon Optimization in Komagataella phaffii.

Dengue is a mosquito-borne viral disease caused by the dengue virus (DENV), which affects millions worldwide. The envelope (E) protein, particularly domain III (EDIII), plays a critical role in viral entry and is a major target for diagnostic and vaccine development. In this study, we aimed to express and purify EDIII from DENV-3 using Komagataella phaffii as an expression system. The recombinant gene was codonoptimized, cloned into the pPICZαA vector, and integrated into the K. phaffii genome. Protein expression was induced with methanol and purified using Ni2+-affinity chromatography, yielding 209-309mg/L. SDS-PAGE and Western blot confirmed protein expression. The recombinant antigen was evaluated in an indirect ELISA with 92 serum samples, demonstrating an AUC of 0.94 (IgM) and 0.828 (IgG), with 92% specificity for IgM. These results suggest that K. phaffii is a suitable host for recombinant dengue antigen production, offering a scalable and cost-effective strategy for diagnostic applications.

Scalable approach for Coronavirus-like particles making based on the Spike protein using Sf9 cells.

Scalable approach for Coronavirus-like particles making based on the Spike protein using Sf9 cells.