Recombinant Protein Production Research Articles

Plant-Based Antigen Production Strategy for SARS-CoV-2 Nucleoprotein and RBD and Its Application for Detection of Antibody Responses in COVID-19 Patients

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the development of efficient serological tests for monitoring the dynamics of the disease as well as the immune response after illness or vaccination was critical. In this regard, low-cost and fast production of immunogenic antigens is essential for the rapid development of diagnostic serological kits. This study assessed the plant-based production of nucleoprotein (N) of SARS-CoV-2 and chimeric receptor-binding domain (RBD) of SARS-CoV-2 presented by hepatitis E virus capsid (HEV/RBD) and validation of the plant-derived proteins as diagnostic antigens for serological tests. The target proteins were expressed in and purified from Nicotiana benthamiana plants. The resulting yield of chimeric HEV/RBD protein reached 100 mg/kg fresh weight and 30 mg/kg fresh weight for N protein. The purified N protein and HEV/RBD protein were used to develop an indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies to SARS-CoV-2 in human sera. To validate the iELISA tests, a panel of 84 sera from patients diagnosed with COVID-19 was used, and the results were compared to those obtained by another commercially available ELISA kit (Dia.Pro D. B., Sesto San Giovanni, Italy). The performance of an HEV/RBD in-house ELISA showed a sensitivity of 89.58% (95% Cl: 75.23–95.37) and a specificity of 94.44% (95% Cl: 76.94–98.2). Double Recognition iELISA based on HEV/RBD and N protein is characterized by a lower sensitivity of 85.42% (95% Cl: 72.24–93.93) and specificity of 94.44% (95% Cl: 81.34–99.32) at cut-off = 0.154, compared with iELISA based on HEV/RBD. Our study confirms that N and fusion HEV/RBD proteins, which are transiently expressed in plants, can be used to detect responses to SARS-CoV-2 in human sera reliably. Our research validates the commercial potential of using plants as an expression system for recombinant protein production and their application as diagnostic reagents for serological detection of infectious diseases, hence lowering the cost of diagnostic kits.

Combined effect of light and magnetic field on recombinant protein production driven by photosystem II-related gene promoters in the cyanobacterium Synechococcus elongatus PCC 7942

Combined effect of light and magnetic field on recombinant protein production driven by photosystem II-related gene promoters in the cyanobacterium Synechococcus elongatus PCC 7942

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.

Screening microorganisms with robust and stable protein expression and secretion capacity.

Robust and stable protein secretion is crucial for efficient recombinant protein production. Here, a novel and powerful platform using split GFP activated droplet sorting (SGADS) has been developed to significantly boost the yields of the protein of interest (POI). The SGADS platform leverages solubilizing peptide P17 and secretory expression in Bacillus subtilis to optimize two split GFP sensors: the P17-GFP1-9/GFP10-POI-GFP11 sensor for assessing protease activity and the P17-GFP1-10/GFP11-POI sensor for measuring secretion capacity. This innovative platform has demonstrated its effectiveness by successfully screening high-performance mutant strains capable of producing collagen, amylase, and protein glutaminase across a range of host organisms, including Escherichia coli, Bacillus subtilis, and Pichia pastoris. The substantial increases in production achieved with the SGADS platform highlight its broad applicability and potential in enhancing recombinant protein production.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes.

T7 RNA polymerase (RNAP) has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5' methyl guanosine caps on T7 RNAP-derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

Efficient production of recombinant hybrid mussel proteins with improved adhesion

Efficient production of recombinant hybrid mussel proteins with improved adhesion

Molecular insights into the aspartate protease Plasmepsin II activity inhibition by fluoroquinolones: A pathway to antimalarial drug development

Plasmepsin II (PlmII) belongs to the aspartate proteases and is involved in hemoglobin degradation in Plasmodium falciparum. Due to its critical role in the survival of the Plasmodium, PlmII is considered as a potent drug target for antimalarial therapy. We have done recombinant protein production of pro-plasmepsin II (Pro-plmII). Pro-PlmII was further activated to mature form (mPlmII) and its activity was confirmed by enzyme kinetics studies. The fluorescence spectroscopic and isothermal titration calorimetric studies show that fluoroquinolone-based antibiotic drugs norfloxacin, ciprofloxacin, and sparfloxacin bind with mPlmII. Molecular docking results show that only norfloxacin and ciprofloxacin are able to bind at the active site of mPlmII via hydrogen binding and hydrophobic interactions. Enzyme kinetics analysis reveals that norfloxacin and ciprofloxacin effectively inhibit mPlmII activity, while sparfloxacin does not exhibit any inhibitory effect on the enzyme's catalytic function. The two methyl groups on the 3rd and 5th carbon atoms of the piperazine ring make sparfloxacin unable to go inside mPlmII and bind at its active site. Overall, the results here suggested that fluoroquinolone-based antibiotic drugs norfloxacin, and ciprofloxacin, can be repurposed as antimalarial inhibitors targeting aspartic proteases. These findings contribute to pave the way for potential therapeutic interventions targeted at malaria.

Expression and purification of Mycobacterium tuberculosis F420-dependent glucose-6-phosphate dehydrogenase enzyme using Escherichia coli.

Since their discovery in Mycobacterium tuberculosis (Mtb), F420-dependent enzymes have been identified as both important drug targets and potential industrial biocatalysts, including for bioremediation of otherwise recalcitrant substrates. Mtb-FGD1, utilizes glucose 6-phosphate (G6P) as an electron donor for the reduction of F420. Current expression systems for Mtb-FGD1 use Mycobacterium smegmatis as host, because of the tendency for it to form inclusion bodies in E. coli. However, large scale recombinant protein production using M. smegmatis is slow and costly and the organism is not generally recognized as safe. Here, we report a faster, cheaper and safer approach for the expression of fully functional Mtb-FGD1 in E. coli using cold-adapted GroEL/ES as chaperones. Our approach yielded ∼ 70 mg of protein per litre (L) of culture. The purified enzyme catalysed the reduction of F420 to F420.H2 in the presence of G6P, and the re-oxidation of the F420.H2 to F420 when coupled to Tfu-FNO, which is a thermostable oxidoreductase that utilizes F420 for the reversible oxidation of NADPH. This latter finding provides opportunity for the utilization of Mtb-FGD1 as an industrial biocatalyst or in the detoxification of environmental contaminants such as malachite green, picrate and aflatoxin.

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.

Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries.

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.

Cell-Based Assays to Detect Innate Immune Response Modulating Impurities: Application to Biosimilar Insulin

Characterizing and mitigating factors that impact product immunogenicity can aid in risk assessment and/or managing risk following manufacturing changes. For follow-on products that have the same indication, patient population, and active product ingredient, the residual immunogenicity risk resides predominantly on differences in product and process related impurities. Characterizing differences in innate immune modulating impurities (IIRMI), which could act as adjuvants by activating local antigen presenting cells (APCs), can inform the immunogenicity risk assessment potentially reducing the need for clinical trials. To date, assays to detect trace levels of IIRMI are being used to support regulatory decisions by FDA for selected synthetic peptide drug products that refer to reference listed drugs of rDNA origin but not recombinant protein or peptide products where more complex mixtures of trace impurities including host cell proteins are expected. Here we describe an exercise to explore whether or not there are differences in the innate immune response elicited by an insulin glargine (produced in E. coli) and its interchangeable biosimilar insulin (produced in P. pastoris) that could indicate differences in IIRMI. Our results suggest the two products elicit comparable innate immune responses as determined by the expression of 90 immune-related genes, including IL-1α, IL-1β, IL-6, CCL3, CCL2, and CXCL8. The data suggest that these assays can provide useful information when assessing recombinant proteins for the presence of IIRMI.Graphical

Virus-like Particles Produced in Plants: A Promising Platform for Recombinant Vaccine Development.

The capsid proteins of many viruses are capable of spontaneous self-assembly into virus-like particles (VLPs), which do not contain the viral genome and are therefore not infectious. VLPs are structurally similar to their parent viruses and are therefore effectively recognized by the immune system and can induce strong humoral and cellular immune responses. The structural features of VLPs make them an attractive platform for the development of potential vaccines and diagnostic tools. Chimeric VLPs can be obtained by attaching foreign peptides to capsid proteins. Chimeric VLPs present multiple copies of the antigen on their surface, thereby increasing the effectiveness of the immune response. Recombinant VLPs can be produced in different expression systems. Plants are promising biofactories for the production of recombinant proteins, including VLPs. The main advantages of plant expression systems are the overall low cost and safety of plant-produced products due to the absence of pathogens common to plants and animals. This review provides an overview of the VLP platform as an approach to developing plant-produced vaccines, focusing on the use of transient expression systems.

Potyvirus-Based Vectors for Heterologous Gene Expression in Plants.

Over the past two decades, plant viral vectors have emerged as a powerful tool for the production of recombinant proteins in plants. Among the different plant viruses engineered to carry foreign genes of interest in their genomes, potyviruses have gained attention due to their polyprotein expression strategy and broad host range. To date, at least eleven different species belonging to the genus Potyvirus have been used for heterologous gene expression in both their natural and experimental hosts. This review article provides an overview of the current state of potyvirus-based plant viral vectors, discussing the advantages and limitations of these systems. We also discuss the future challenges and potential applications of potyvirus-based expression vectors, including the production of vaccines, nanoparticles, therapeutics, and metabolic engineering. Overall, we highlight the potential of potyvirus-based vectors as a versatile tool for recombinant protein production in plants.

Significant Enhancement Catalytic Activity of Nitrile Hydratase by Balancing the Subunits Expression.

Escherichia coli (E. coli) is the most commonly used bacterial recombinant protein production system due to its easy genetic modification properties. In our previous study, a recombinant plasmid expressing the Fe-type nitrile hydratase derived from Rhodococcus erythropolis CCM2595 (ReNHase) was successfully constructed and the recombinant ReNHase exerted an excellent catalytic effect on dinitrile compounds. Nevertheless, the ReNHases were confronted with imbalanced subunit expression during heterologous expression, which restricted the enzymes assemble functionally. In this study, the secondary structure of mRNA in the ribosome binding sequence region of the β-subunit was optimized to elevate the translation efficiency of the β-subunit gene and balance the expression of α- and β-subunits in ReNHase. The optimized ReNHase showed a 12-fold increase in specific enzyme activity over wild-type ReNHase. To further enhance the soluble expression of ReNHase, the ReNHase was labeled using three different fusion tags, resulting in three new recombinant ReNHases. In these recombinant ReNHases, some of the fusion tags promoted the soluble expression of ReNHase, but also affected the balance of α-/β-subunit expression and the secondary structure of the ReNHase, and reduced the enzyme activity. In conclusion, our results provide an optimized strategy for the heterologous expression of multi-subunit proteins.

Production of recombinant protein of Tyr p 32 from Tyrophagus putrescentiae and identifying its immunoreactivity

The present study was aimed to produce the recombinant protein of Tyrophagus putrescentiae allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of T. putrescentiae and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells. After being induced with IPTG, the recombinant protein was purified with Ni column, and then identified on SDS-PAGE and Western blotting. Serum of children with allergic asthma and(or) rhinitis was collected, IgE-ELISA and IgE-Western blotting were used to detect the binding rate of rTyr p 32 to human T.putrescentiae-positive serum. After human bronchial epithelial cells BEAS-2B being cultured with rTyr p 32, the expression levels of IL-6 and IL-8 cytokines was detected by ELISA and qRT-PCR, t-test was used for pairwise comparison between groups. The results showed that the cDNA length of Tyr p 32 was 885 bp. The sequence identity between Tyr p 32 and Der p 32, Der f 32 was 70.21% and 68.03%, respectively. According to SDS-PAGE and Western blotting, the molecular weight of rTyr p 32 was about 35 000 Da, which was consistent with the theoretical value. IgE-ELISA results showed that the positive rate of rTyr p 32 was 41.38% (12/29) against T. putrescentiae-positive serum. When BEAS-2B cells were cultured with rTyr p 32, the expression of IL-6 and IL-8 increased in the cell supernatant in a dose-dependent manner (t=-29.10,P=0.001 2;t=-33.69,P=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 (t=-9.15,P=0.011 7;t=-17.16,P=0.003 4). In conclusion, the recombinant protein rTyr p 32 was successfully prepared, which provides raw materials for component diagnosis and specific immunotherapy of allergic diseases.

A UHPLC-QE-MS-based metabolomics approach for the evaluation of fermented lipase by an engineered Escherichia coli

Using an engineered Escherichia coli to produce lipase and can easily achieve high-level expression. The investigation of biochemical processes during lipase fermentation, approached from a metabolomics perspective, will yield novel insights into the efficient secretion of recombinant proteins. In this study, the lipase batch fermentation was carried out first with enzyme activity of 36.83 U/mg cells. Then, differential metabolites and metabolic pathways were identified using an untargeted metabolomics approach through comparative analysis of various fermentation periods. In total, 574 metabolites were identified: 545 were up-regulated and 29 were down-regulated, mainly in 153 organic acids and derivatives, 160 organoheterocyclic compounds, 64 lipids and lipid-like molecules, and 58 organic oxygen compounds. Through metabolic pathways and network analysis, it could be found that tryptophan metabolism was of great significance to lipase production, which could affect the secretion and synthesis of recombinant protein. In addition, the promotion effects of cell growth by varying concentrations of indole acetic acid serve to validate the results obtained from tryptophan metabolism. This study offers valuable insights into metabolic regulation of engineered E. coli, indicating that its fermentation bioprocess can be systematically designed according to metabolomics findings to enhance recombinant protein production.

Continuous Cultivation of Yarrowia lipolytica: Potential, Challenges, and Case Studies

Currently, the yeast Yarrowia lipolytica is regarded as one of the most promising producers of protein, lipids, polyols, organic acids, and other metabolites. The objective of enhancing the efficiency of the target product biosynthesis can be achieved through the improvement of the strains-producers and the optimization of the cultivation conditions. The present review assesses the potential of continuous cultivation methods (chemostat, turbidostat, pH-auxostat, changestats, etc.) in order to gain insight into the impact of strains and cultivation conditions on the productivity of the developed bioprocesses. The utilization of continuous cultivation methods enables the implementation of processes under controlled and reproducible conditions, thus stabilizing the parameters of the cultivation and the physiological state of the producer, and obtaining homogeneous samples. The review focuses on nitrogen-limited chemostat cultures, which represent the most commonly employed strategy for investigating the physiological and biochemical characteristics of the yeast Y. lipolytica and for developing the processes for the production of lipids, erythritol, citric acid, and recombinant proteins. To date, such an analysis of the literature has not been conducted in the context of the yeast Y. lipolytica.

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.

Creating a System of Dual Regulation of Translation and Transcription to Enhance the Production of Recombinant Protein.

When constructing cell factories, it is crucial to reallocate intracellular resources towards the synthesis of target compounds. However, imbalanced resource allocation can lead to a tradeoff between cell growth and production, reducing overall efficiency. Reliable gene expression regulation tools are needed to coordinate cell growth and production effectively. The orthogonal translation system, developed based on genetic code expansion (GCE), incorporates non-canonical amino acids (ncAAs) into proteins by assigning them to expanded codons, which enables the control of target protein expression at the translational level in an ncAA-dependent manner. However, the stringency of this regulatory tool remains inadequate. This study achieved strict translational-level control of the orthogonal translation system by addressing the abnormal leakage caused by the arabinose-inducible promoter. Further validation was conducted on the relationship between ncAA concentration and expression level, as well as the host's adaptability to the system. Subsequently, the system's applicability across multiple Escherichia coli hosts was verified by examining the roles of RF1 (peptide chain release factor 1) and endogenous TAG codons. By combining this strategy with inducible promoters, dual-level regulation of target gene expression at both transcriptional and translational levels was achieved and the dynamic range was further increased to over 20-fold. When using ncAA to control the expression of T7 RNA polymerase (T7 RNAP), the leakage expression was reduced by 82.7%, mitigating the low production efficiency caused by extensive leakage in the T7 system. As proof of concept, the strategy enhanced the production of alcohol dehydrogenase (ADH) by 9.82-fold, demonstrating its excellent capability in controlling gene expression in developing cell factories.