RESUMO
IMPORTANCE: Flavonoids are a group of compounds generally produced by plants with proven biological activity, which have recently beeen recommended for the treatment and prevention of diseases and ailments with diverse causes. In this study, naringenin was produced in adequate amounts in yeast after in silico design. The four genes of the involved enzymes from several organisms (bacteria and plants) were multi-expressed in two vectors carrying each two genes linked by a short viral peptide sequence. The batch kinetic behavior of the product, substrate, and biomass was described at lab scale. The engineered strain might be used in a more affordable and viable bioprocess for industrial naringenin procurement.
Assuntos
Flavanonas , Flavonoides , Flavonoides/metabolismo , Saccharomyces cerevisiae/metabolismo , Flavanonas/metabolismoRESUMO
Cyclin/cyclin-dependent kinase (CDK) heterodimers have multiple phosphorylation targets and may alter the activity of these targets. Proteins from different metabolic processes are among the phosphorylation targets, that is, enzymes of central carbon metabolism. This work explores the interaction of Cyc/CDK complex members with the glycolytic enzymes hexokinase 7 (HXK7) and glyceraldehyde-3-phosphate dehydrogenase (GAP). Both enzymes interacted steadily with CycD2;2, CycB2;1 and CDKA;1 but not with CDKB1;1. However, Cyc/CDKB1;1 complexes phosphorylated both enzymes, decreasing their activities. Treatment with a CDK-specific inhibitor (RO-3306) or with lambda phosphatase after kinase assay restored total HXK7 activity, but not GAP activity. In enzymatic assays, increasing concentrations of CDKB1;1, but not of CycD2;2, CycB2;1 or CycD2;2/CDKB1;1 complex, decreased GAP activity. Cell cycle regulators may modulate carbon channeling in glycolysis by two different mechanisms: Cyc/CDK-mediated phosphorylation of targets (e.g., HXK7; canonical mechanism) or by direct and transient interaction of the metabolic enzyme (e.g., GAP) with CDKB1;1 without a Cyc partner (alternative mechanism).
Assuntos
Proteínas de Ciclo Celular , Hexoquinase , Proteínas de Ciclo Celular/metabolismo , Zea mays/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Ciclo CelularRESUMO
The progression of SARS-CoV-2 (COVID-19) in humans heavily depends on the patient's overall health status, especially on its immunoregulatory capacity. Different plants and plant-derived preparations (infusions, encapsulated, etc.) have been used as immunoregulators, several of them with scientific support. Nevertheless, due to the composition complexity of such plant-derived preparations, the molecular and physiological mechanisms involved in their beneficial effects remain, in some cases, unclear. In this review article, the most reported plants used in traditional medicine to enhance immunoregulatory capacity are presented, and their effect on the innate immune response is discussed and correlated with their respective phytochemical profile. Understanding how the plant phytochemical profile relates to the observed impact on the innate and adaptative immune response is fundamental to designing plant-derived co-treatments to lessen the symptoms and favor the recovery of COVID-19 patients. In this regard, we propose a prospective guideline for using plants and plant-derived preparations as co-treatments for COVID-19 (and similar viral infections), which could be helpful in the context of the worldwide effort to end the current SARS-CoV-2 pandemic.
Assuntos
COVID-19 , Plantas Medicinais , Humanos , Plantas Medicinais/química , SARS-CoV-2 , Estudos Prospectivos , Medicina Tradicional , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/uso terapêutico , ImunidadeRESUMO
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Bactérias , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Transporte de Cátions , Proteínas de Escherichia coli , Escherichia coli , Synechocystis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/isolamento & purificação , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Transporte de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Synechocystis/metabolismoRESUMO
Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.
RESUMO
Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cobre , Escherichia coli/genética , Proteínas de Escherichia coli/química , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histidina/química , Metais/química , Proteínas Recombinantes de Fusão/genética , SolubilidadeRESUMO
Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.