Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

Enhanced prokaryotic expression, purification, and biological activities of human keratinocyte growth factor.

Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), plays a critical role in embryonic development, cell proliferation, and differentiation. However, efficient production of recombinant KGF remains a challenge due to its low expression levels and high tendency for aggregation in Escherichia coli. This study aimed to enhance the expression and solubility of KGF by employing different protein tags-PDIb'a', MBP, and His-fused to the N-terminus of KGF. Among these, H-PDIb'a'-KGF demonstrated superior stability and was selected for large-scale production and purification. The purified KGF was confirmed through liquid chromatography with tandem mass spectrometry analysis, which showed an 81% fragment mass identification coverage. Biological activity assessments using human breast cancer MCF-7 cells indicated that purified KGF significantly increased cell proliferation, with an EC50 of 6.4 ± 0.5 pM. Interestingly, PDIb'a' alone also exhibited a stimulatory effect on MCF-7 cells. Furthermore, the purified KGF enhanced the wound healing of HaCaT keratinocytes in a dose-dependent manner. These findings provide valuable insights into the efficient production and functional characterization of recombinant KGF for potential applications in therapeutic interventions.

Anti-inflammatory effect of Piper longum L. fruit methanolic extract on lipopolysaccharide-treated RAW 264.7 murine macrophages.

Context: The Piper species was studied several potential properties such as anti-tumor, anti-inflammatory and antioxidant activity. However, the specific anti-inflammatory activity of the extract from the fruits of P. longum L. has not been investigated. Objectives: Our study want to examine the anti-inflammatory effects of P. longum L. fruit methanolic extracts (PLE) on lipopolysachharide (LPS)-stimulated RAW 264.7 murine macrophages to understand the mechanism of this effect. Method: This study examined the chemical profiling of PLE by LC-HRMS analysis and measured the presence of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the supernatant using the Griess reagent assay and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA expression of IL-6, TNF-α, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were evaluated by using real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, the protein expression of COX-2, iNOS and the phosphorylation of MAPK family, c-Jun N-terminal kinase (JNK), p38 in protein level were observed by western blotting. Result: PLE have detected 66 compounds which belong to different classes such as alkaloids, flavonoids, terpenoids, phenolics, lactones, and organic acids inhibited nitric oxide products with the IC50 = 28.5 ± 0.91 µg/mL. Moreover, PLE at 10-100 µg/mL up-regulate HO-1 protein expression from 3 to 10 folds at 3 h. It also downregulated the mRNA and protein expression of iNOS, COX-2, decreased IL-6 and TNF-α secretion by modulating the mitogen-activated protein kinase (MAPK) signaling pathway, specifically by decreasing the phosphorylation of p38 and JNK. Conclusion: These results shown chemical profiling of PLE and demonstrated that PLE exhibits anti-inflammatory effects by regulating the MAPK family and could be a potential candidate for the treatment of inflammatory diseases.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Efflux Pump Inhibitors in Controlling Antibiotic Resistance: Outlook under a Heavy Metal Contamination Context.

Multi-drug resistance to antibiotics represents a growing challenge in treating infectious diseases. Outside the hospital, bacteria with the multi-drug resistance (MDR) phenotype have an increased prevalence in anthropized environments, thus implying that chemical stresses, such as metals, hydrocarbons, organic compounds, etc., are the source of such resistance. There is a developing hypothesis regarding the role of metal contamination in terrestrial and aquatic environments as a selective agent in the proliferation of antibiotic resistance caused by the co-selection of antibiotic and metal resistance genes carried by transmissible plasmids and/or associated with transposons. Efflux pumps are also known to be involved in either antibiotic or metal resistance. In order to deal with these situations, microorganisms use an effective strategy that includes a range of expressions based on biochemical and genetic mechanisms. The data from numerous studies suggest that heavy metal contamination could affect the dissemination of antibiotic-resistant genes. Environmental pollution caused by anthropogenic activities could lead to mutagenesis based on the synergy between antibiotic efficacy and the acquired resistance mechanism under stressors. Moreover, the acquired resistance includes plasmid-encoded specific efflux pumps. Soil microbiomes have been reported as reservoirs of resistance genes that are available for exchange with pathogenic bacteria. Importantly, metal-contaminated soil is a selective agent that proliferates antibiotic resistance through efflux pumps. Thus, the use of multi-drug efflux pump inhibitors (EPIs) originating from natural plants or synthetic compounds is a promising approach for restoring the efficacy of existing antibiotics, even though they face a lot of challenges.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Development of a Pteris vittata L. compound database by widely targeted metabolomics profiling.

The objective of this work was the development of a detailed, extensive and reliable database of the metabolomes of P. vittata. Using an ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry system (UPLC-QqQ-MS/MS) and based on the knowledge of retention time and mass spectral characteristics of an in-house collection of authentic standards, we screened for the presence of a large collection of natural compounds. The database represents 359 authenticated metabolites, comprising 220 primary and 139 secondary metabolites (70 flavonoids, 16 phenylpropanoic acid derivatives, five coumarins, two stilbenoids, 14 benzoic acids, nine phenols, 20 alkaloids and three terpenoids). Comparison of the accumulation of these compounds in two tissues showed that the aerial parts were enriched in flavonols, whereas the subterranean parts were enriched in anthocyanins. The comprehensive database developed here will be beneficial in improving the understanding of the chemical basis of plant therapeutic profile using multivariate analysis, with a particular example of antioxidant activity.

Metabolic markers for the yield of lipophilic indole alkaloids in dried woad leaves (Isatis tinctoria L.).

The pharmacologically active dichloromethane extracts of dried woad leaves (Isatis tinctoria L.), and the methanol extracts of comparable fresh leaves of the same plants, were analyzed by LC-MSn. The fresh leaf metabolite profile revealed a complex pattern of indolic compounds. Besides the known indigo precursors, isatan A, isatan B and indican, seven previously unreported indole derivatives were characterized: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside, 6-hydroxyindole-3-carboxylic acid 6-O-glucoside and 6-hydroxyindole-3-carboxylic acid glucose ester. The integration of 122 compounds in fresh leaves and of five selected compounds (indoxyl, isatin, indigo, indirubin, and tryptanthrin) in dried leaves, formed the input data for a stepwise modelling procedure generating five predictive linear models. The structure of the predictive models and a cross validation provide evidence that the models could predict well or moderately well the accumulation of the selected lipophilic compounds, and were simple enough to be used in a woad cultivation program. PLS regression models relating each of the five selected dry leaf indolics to the fresh leaf metabolome were then fitted in order to deduct potential precursors and mechanisms leading to the formation of these lipophilic indolics in drying woad leaves. The models suggested glucobrassicin, isatan A and isatan B as the main candidate precursors of these compounds, besides a minor contribution of other fresh leaf indolics, including malonylindican, actylindican and dioxindole malonylglucoside. Dioxindole malonylglucoside was identified here as isatan C. The models further suggested that the accumulation of phenylpropanoid antioxidants in woad leaves has a negative impact on the formation of indoxyl, isatin, indigo, indirubin and tryptanthrin.

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism.

BACKGROUND: The production of secondary metabolites through the culture of entire plants is of great interest. Soilless culture, such as hydroponics, enables the control of plant growth and metabolism. Specific environmental conditions must be developed to maximize the productivity of medicinal plants used as efficient natural bioreactors. METHODS: The nutrient solution of newly established hydroponic cultures ofDatura innoxia Mill. were inoculated with Agrobacterium rhizogenes (A.r.) wild strains (TR7, TR107, 11325 or 15834). Growth and the alkaloid contents of roots and aerial parts were analyzed. Axenic cultures were also performed with modified TR7 strains containing the egfp or gus reporter gene. In vitro isolated root cultures enabled the phenological and molecular demonstration of gene transfer. RESULTS: A.r.TR 7 led to a greater improvement in plant secondary metabolism and growth. Positive expression of the reporter genes occurred. Isolation and subculture of some of the roots of these plants showed a hairy root phenotype; molecular tests proved the transfer of bacterial genes into the roots isolated from the plants. CONCLUSIONS: Hyoscyamine and scopolamine productivity is enhanced after A.r. inoculation in the nutrient solution of hydroponic plants. Transformation events occur in the original roots of the plants. This leads to chimeric plants with a part of their roots harboring a hairy root phenotype. Such semi-composite plants could be used for successful specialized metabolite bioproduction in greenhouses.

Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves.

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

Impact of metal stress on the production of secondary metabolites in Pteris vittata L. and associated rhizosphere bacterial communities.

Plants adapt to metal stress by modifying their metabolism including the production of secondary metabolites in plant tissues. Such changes may impact the diversity and functions of plant associated microbial communities. Our study aimed to evaluate the influence of metals on the secondary metabolism of plants and the indirect impact on rhizosphere bacterial communities. We then compared the secondary metabolites of the hyperaccumulator Pteris vittata L. collected from a contaminated mining site to a non-contaminated site in Vietnam and identified the discriminant metabolites. Our data showed a significant increase in chlorogenic acid derivatives and A-type procyanidin in plant roots at the contaminated site. We hypothesized that the intensive production of these compounds could be part of the antioxidant defense mechanism in response to metals. In parallel, the structure and diversity of bulk soil and rhizosphere communities was studied using high-throughput sequencing. The results showed strong differences in bacterial composition, characterized by the dominance of Proteobacteria and Nitrospira in the contaminated bulk soil, and the enrichment of some potential human pathogens, i.e., Acinetobacter, Mycobacterium, and Cupriavidus in P. vittata's rhizosphere at the mining site. Overall, metal pollution modified the production of P. vittata secondary metabolites and altered the diversity and structure of bacterial communities. Further investigations are needed to understand whether the plant recruits specific bacteria to adapt to metal stress.

Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks.

The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected.