scholarly journals Plasmodium falciparum CTP:phosphocholine cytidylyltransferase expressed in Escherichia coli: purification, characterization and lipid regulation

1997 ◽  
Vol 324 (3) ◽  
pp. 903-910 ◽  
Author(s):  
Hye-Jeong YEO ◽  
Marie-Pierre LARVOR ◽  
Marie-Laure ANCELIN ◽  
Henri J. VIAL
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
Oleic Acid ◽  
Recombinant Protein ◽  
Neutral Lipids ◽  
Insoluble Fraction ◽  
Biochemical Properties ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Lipid Regulation

The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) has been isolated from an overexpressing strain of Escherichia coli. The plasmid pETPfCCT mediated the overexpression of the full-length polypeptide directly. The recombinant protein corresponded to 6–9% of the total cellular proteins and was found essentially in the insoluble membrane fraction. Urea at 6 M was used to solubilize the recombinant protein from the insoluble fraction. The CCT activity was restored upon the removal of urea, and the protein was subsequently purified to homogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, including in vitro substrate specificity and enzymic characterization, were assessed. The lipid regulation of the recombinant plasmodial CCT activity was characterized for the first time. The Km values were 0.49±0.03 mM (mean±S.E.M.) for phosphocholine and 10.9±0.5 mM for CTP in the presence of lipid activators (oleic acid/egg phosphatidylcholine vesicles), whereas the Km values were 0.66±0.07 mM for phosphocholine and 28.9±0.8 mM for CTP in the absence of lipid activators. The PfCCT activity was stimulated to the same extent in response to egg phosphatidylcholine vesicles containing anionic lipids, such as oleic acid, cardiolipin and phosphatidylglycerol, and was insensitive or slightly sensitive to PC vesicles containing neutral lipids, such as diacylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme activity by oleic acid was antagonized by the cationic aminolipid sphingosine. These lipid-dependence properties place the parasite enzyme intermediately between the mammalian enzymes and the yeast enzyme.

Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

2018 ◽  
Vol 18 (4) ◽  
pp. 34-41
Author(s):  
Sergey A. Ishuk ◽  
Elena G. Bogomolova ◽  
Olga A. Dobrovolskaya ◽  
Alyona O. Akhmetshina ◽  
Daria S. Krasnoshchek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Cation Exchange ◽  
Expression System ◽  
Neuroblastoma Cells ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Recombinant Insulin ◽  
Prokaryotic Expression System

This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein. To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography. The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons. Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.

scholarly journals Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

2007 ◽  
Vol 408 (2) ◽  
pp. 173-180 ◽  
Author(s):  
Paul W. Bowyer ◽  
Ruwani S. Gunaratne ◽  
Munira Grainger ◽  
Chrislaine Withers-Martinez ◽  
Sasala R. Wickramsinghe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Scale ◽  
Expression And Purification ◽  
Purification System ◽  
Pure Protein ◽  
Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

scholarly journals Sistemas de expresión de proteínas recombinantes para el análisis funcional de antígenos de Plasmodium falciparum y Plasmodium vivax: una revisión

2021 ◽  
Vol 8 (2) ◽  
Author(s):  
Alida Marcela Gómez Rodríguez ◽  
Laura Esperanza Cuy-Chaparro ◽  
Anny Jineth Camargo Marcipe
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
Plasmodium Vivax

Introducción. Comprender la función de antígenos de Plasmodium spp., involucrados en invasión a células hospederas es necesario para diseñar vacunas. Diferentes estudios han generado proteínas recombinantes utilizando sistemas de expresión heterólogos, obteniendo moléculas semejantes a las nativas. Estos avances son fundamentales para desarrollar estrategias que bloqueen la infección de estos patógenos. Objetivo. Describir las características y aspectos metodológicos más importantes de sistemas de expresión de proteínas recombinantes en estudios funcionales de Plasmodium spp. Metodoogía. Revisión descriptiva de estudios publicados en PubMed, Science Direct, Embase y MedLine. Criterios de inclusión: trabajos publicados entre el 2010 y 2020 que incluyeran sistemas recombinantes en células de Escherichia coli, de mamífero y libre de células, para estudios funcionales de antígenos de Plasmodium falciparum y Plasmodium vivax. Se revisaron 70 artículos originales, 58 cumplieron con los criterios establecidos y 12 utilizaron otros sistemas heterólogos diferentes a los analizados en este estudio.    Resultados. Obtener proteínas recombinantes mediante sistema procariota, de mayor rendimiento y bajo costo, ha permitido estudios funcionales de un número importante de antígenos. Los sistemas con células de mamífero y libre de células, que permiten modificaciones pos-traduccionales y plegamiento adecuado de moléculas, se usan para producir librerías de antígenos con estructura conformacional similar a la nativa.  Conclusión. Estudio de antígenos de Plasmodium spp. implicados en infección y desarrollo en células diana, requiere adecuada selección del método de producción recombinante. El refinamiento de procesos de expresión en sistemas procariotas, eucariotas e in vitro, mediante ingeniería genética y cultivo celular, permitirá mejores rendimientos y menor costo.

scholarly journals Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.

1985 ◽  
Vol 5 (6) ◽  
pp. 1449-1455 ◽  
Author(s):  
S Hattori ◽  
L S Ulsh ◽  
K Halliday ◽  
T Y Shih
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Active Center ◽  
Biochemical Properties ◽  
Scatchard Analysis ◽  
Single Class ◽  
P21 Protein ◽  
High Level

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

scholarly journals The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System inRhizorhabdus dicambivoransNdbn-20

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Shigang Yao ◽  
Le Chen ◽  
Zhou Yang ◽  
Li Yao ◽  
Jianchun Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Reductase Activity ◽  
Gene Clusters ◽  
Biochemical Properties ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Metabolic Genes ◽  
Methyltransferase Gene

ABSTRACTThe herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system inRhizorhabdus dicambivoransNdbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely,metF(coding for 5,10-CH2-THF reductase),folD(coding for 5,10-CH2-THF dehydrogenase–5,10-methenyl-THF cyclohydrolase), andpurU(coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. ThemetFgene of scaffold 66 (metF1) was expressed inEscherichia coliBL21(DE3), and the product was purified as a His6-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH3-THF dehydrogenase activityin vitro. ThekcatandKmfor 5-CH3-THF were 0.23 s−1and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed thatmetF1is essential for dicamba degradation, whereasfolD1is dispensable.IMPORTANCEThere are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome ofR. dicambivoransNdbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF-dependent dicamba demethylation system inR. dicambivoransNdbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1in vitrodid not show 5,10-CH2-THF reductase activity, which is the physiological function ofEscherichia coliMetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.

scholarly journals Cytoadherence by Plasmodium falciparum-infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes.

1988 ◽  
Vol 168 (4) ◽  
pp. 1307-1320 ◽  
Author(s):  
C Magowan ◽  
W Wollish ◽  
L Anderson ◽  
J Leech
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Molecular Size ◽  
Melanoma Cells ◽  
Biochemical Properties ◽  
Surface Proteins ◽  
In Vitro Techniques ◽  
Molecular Sizes ◽  
Triton X

Plasmodium falciparum-infected erythrocytes (IRBCs) adhere specifically to venular endothelium and thereby evade spleen-dependent immune mechanisms. We have investigated the molecular basis of cytoadherence. We report here that the capacity for cytoadherence of IRBCs is correlated with the expression of a family of variable proteins on the surface of IRBCs. Essential to these studies was the use of in vitro techniques for modulating the cytoadherence phenotype of cloned parasites. In initial studies, we found culture-adapted parasites to be poorly cytoadherent or noncytoadherent. To select for cytoadherent parasites, we incubated knobbed IRBCs with C32 melanoma cells and cultured the adherent cells. Repeated rounds of selection produced parasites with increased cytoadherence. To select for noncytoadherent parasites, we cultured the cells that did not adhere to C32 melanoma cells. Cytoadherent IRBCs from two different cloned isolates had large (Mr greater than 2.4 x 10(5) radioiodinatable proteins that differed in size between the isolates but had in common the biochemical properties of trypsin sensitivity and insolubility with Triton X-100. The proteins were not detected with uninfected erythrocytes, indicating that they were parasite determined, nor were they detected with IRBCs containing parasites cultured for many months without selection. With continued selection for the cytoadherent phenotype, additional IRBC surface proteins with larger molecular sizes (Mr 2.9 x 10(5) and 3.2 x 10(5] appeared. A sequence of reversible changes in the cytoadherence phenotype of cloned parasites was accompanied by variation in the molecular size of the IRBC surface protein. Increased cytoadherence was correlated with expression of larger proteins and decreased cytoadherence was correlated with expression of smaller proteins; there was no change in the molecular size of two other parasite proteins associated with the IRBC membrane. The results indicate that the expression of this family of proteins is closely linked to the cytoadherence phenotype of the parasites, suggesting that the members of the protein family have a role in mediating cytoadherence between IRBCs and endothelial cells.

scholarly journals Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

2020 ◽  
Vol 21 (3) ◽  
pp. 928 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yulia A. Logashina ◽  
Yaroslav A. Andreev
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Reaction Medium ◽  
Cellular Metabolism ◽  
Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

scholarly journals A Plasmodium falciparum lysophospholipase regulates fatty acid acquisition for membrane biogenesis to enable schizogonic asexual division

2021 ◽  
Author(s):  
Pradeep Kumar Sheokand ◽  
Yoshiki Yamaryo-Botte ◽  
Vandana Thakur ◽  
Mudassir M Banday ◽  
Mohd Asad ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutral Lipids ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Phospholipid Metabolism ◽  
Parasite Development ◽  
Parasite Line ◽  
Membrane Biogenesis ◽  
Drug Candidates

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis during the intracellular life cycle of Plasmodium falciparum. To generate large amounts of phospholipids required during blood stages, the parasite massively scavenge, recycle and reassemble host lipids. P. falciparum possesses an unusual large number of lysophospholipases. However, their functional roles and importance remain to be elucidated. Here, we functionally characterized one of P. falciparum lysophospholipase (PfLPL3) (Gene ID PF3D7_1476800), to reveal its critical role in parasite propagation during asexual blood stages. We generated a transgenic parasite line using GFP-glmS C-terminal tagging approach, for localization as well as inducible knockdown of PfLPL3. PfLPL3 displayed a dynamic localization throughout asexual stages, mainly localizing in the host parasite interface: parasitophorous vacuole space and expanding into the tubulovesicular network within the host cell. Inducible knock-down of PfLPL3 hindered normal intraerythrocytic cycle, specifically causing disruption in parasite development from trophozoites to schizont, as well as reduction in number of merozoites progenies. Thus, down-regulation of PfLPL3 significantly inhibited parasite growth suggesting its critical role for proper parasite propagation during blood stages. Detailed lipidomic analyses showed that PfLPL3 generates fatty-acids for the synthesis of neutral lipids DAG and TAG, whilst controlling the timely synthesis of phospholipids that are crucial for membrane biogenesis required for merozoite development during asexual cycle. Setting up an in vitro activity based screening of Malaria Box allowed identification of specific inhibitors of PfLPL3 having potent parasitical efficacies. These compounds are pertinent both as anti-malarial drug candidates and chemical tools specifically targeting membrane biogenesis during asexual blood stages.

Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody

1985 ◽  
Vol 5 (6) ◽  
pp. 1449-1455
Author(s):  
S Hattori ◽  
L S Ulsh ◽  
K Halliday ◽  
T Y Shih
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Active Center ◽  
Biochemical Properties ◽  
Scatchard Analysis ◽  
Single Class ◽  
P21 Protein ◽  
High Level

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

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