scholarly journals Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor

Marine Drugs ◽  
2019 ◽  
Vol 17 (9) ◽  
pp. 511
Author(s):  
Giovanni Covaleda-Cortés ◽  
Martha Hernández ◽  
Sebastián Alejandro Trejo ◽  
Manuel Mansur ◽  
Sergi Rodríguez-Calado ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Function Analysis ◽  
High Yield ◽  
Marine Snail ◽  
Carboxypeptidase B ◽  
Recombinant Production ◽  
Disulphide Bonds ◽  
Proteinaceous Inhibitor ◽  
Direct Applicability ◽  
Twofold Decrease

A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.

scholarly journals High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Axel Abelein ◽  
Gefei Chen ◽  
Kristīne Kitoka ◽  
Rihards Aleksis ◽  
Filips Oleskovs ◽  
...  
Keyword(s):  
Spider Silk ◽  
Amyloid Β ◽  
Silk Gland ◽  
High Yield ◽  
Efficient Production ◽  
Amyloid Β Peptide ◽  
Nephila Clavipes ◽  
Recombinant Production ◽  
Yield Production ◽  
High Yields

AbstractDuring storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.

scholarly journals Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract Background Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. Results A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. Conclusion We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

scholarly journals Characterization of a New Glyoxal Oxidase from the Thermophilic Fungus Myceliophthora thermophila M77: Hydrogen Peroxide Production Retained in 5-Hydroxymethylfurfural Oxidation

Catalysts ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 476 ◽  
Author(s):  
Marco Kadowaki ◽  
Mariana Godoy ◽  
Patricia Kumagai ◽  
Antonio Costa-Filho ◽  
Andrew Mort ◽  
...  
Keyword(s):  
Function Analysis ◽  
Spatial Arrangement ◽  
Oxidative Enzymes ◽  
Lignocellulose Degradation ◽  
Paramagnetic Resonance ◽  
Recombinant Production ◽  
X Ray Scattering ◽  
Cazy Database ◽  
Fermentation Inhibitor ◽  
Glyoxal Oxidase

Myceliophthora thermophyla is a thermophilic industrially relevant fungus that secretes an assortment of hydrolytic and oxidative enzymes for lignocellulose degradation. Among them is glyoxal oxidase (MtGLOx), an extracellular oxidoreductase that oxidizes several aldehydes and α-hydroxy carbonyl substrates coupled to the reduction of O2 to H2O2. This copper metalloprotein belongs to a class of enzymes called radical copper oxidases (CRO) and to the “auxiliary activities” subfamily AA5_1 that is based on the Carbohydrate-Active enZYmes (CAZy) database. Only a few members of this family have been characterized to date. Here, we report the recombinant production, characterization, and structure-function analysis of MtGLOx. Electron Paramagnetic Resonance (EPR) spectroscopy confirmed MtGLOx to be a radical-coupled copper complex and small angle X-ray scattering (SAXS) revealed an extended spatial arrangement of the catalytic and four N-terminal WSC domains. Furthermore, we demonstrate that methylglyoxal and 5-hydroxymethylfurfural (HMF), a fermentation inhibitor, are substrates for the enzyme.

Physical properties of the oleoresin system of the four major southern pines

1977 ◽  
Vol 7 (3) ◽  
pp. 520-525 ◽  
Author(s):  
John D. Hodges ◽  
William W. Elam ◽  
William F. Watson
Keyword(s):  
Physical Properties ◽  
Function Analysis ◽  
Total Yield ◽  
Morphological Characteristics ◽  
High Viscosity ◽  
High Rate ◽  
High Yield ◽  
Breast Height ◽  
Low Viscosity ◽  
Very High

Oleoresin viscosity, flow (rate, duration, and total amount), and rate of crystallization were determined for Pinuselliottii Engelm., Pinuspalustris Mill., Pinustaeda L., and Pinusechinata Mill, in central Louisiana, U.S.A. Physical properties of the oleoresin and tree morphological characteristics (diameter at breast height, growth rate, height, crown ratio, and age) were not strongly related in either of the four species. Pinuselliottii oleoresin was extremenly viscous, crystallized very slowly, and flowed at a slow rate over a long period, and total yield was moderate. Pinuspalustris oleoresin was of moderately high viscosity and very high yield and had a high rate of flow. Pinustaeda and Pinusechinata oleoresin had, on the average, low viscosity, a moderate to low total yield, a short duration of flow, and rapid rate of crystallization. A discriminant function analysis revealed that 19% of the Pinustaeda and 6% of the Pinusechinata trees had oleoresin properties more similar to Pinuspalustris and Pinuselliottii than to the means for their own species. This information is being used to assess tree susceptibility to attack by Dendroctonusfrontalis Zimm.

High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

2015 ◽  
Vol 108 ◽  
pp. 1-8 ◽  
Author(s):  
Christian Zerfaß ◽  
Sandra Braukmann ◽  
Sandor Nietzsche ◽  
Stephan Hobe ◽  
Harald Paulsen
Keyword(s):  
High Yield ◽  
Recombinant Production ◽  
Self Assembling ◽  
Polycationic Peptide

scholarly journals Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

2020 ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract Background: Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or Duet series.Results: A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. Conclusion: We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the Duet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

scholarly journals Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

2020 ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract Background: Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or Duet series.Results: A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. Conclusion: We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the Duet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

scholarly journals Cytotoxicity of Frutalin on Distinct Cancer Cells Is Independent of Its Glycosylation

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4712
Author(s):  
Carla Oliveira ◽  
Ana Isabel Freitas ◽  
Nair Campos ◽  
Lucília Saraiva ◽  
Lucília Domingues
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Active Form ◽  
High Yield ◽  
Human Cancer Cells ◽  
Proliferative Effect ◽  
Recombinant Production ◽  
Tetrameric Structure ◽  
Spectroscopy Studies ◽  
Purification Protocol

Frutalin is a plant lectin with beneficial immunobiological action, although the access to its active form is still restricted. Moreover, there is a knowledge gap on isoform activity and glycosylation impact on its bioactivity, and recombinant production protocols were seen as ineffective. Here, a simpler and faster production and purification protocol was developed, attaining a yield of purified frutalin 3.3-fold higher than that obtained previously. Hemagglutination assays confirmed that this frutalin isoform could not agglutinate rabbit erythrocytes, while maintaining the native tetrameric structure, as indicated by DLS analysis, and strong interaction with methyl-alpha-galactose, in fluorescence spectroscopy studies. The cytotoxicity of the recombinant frutalin isoform was shown in a broad panel of human cancer cells: colon (HCT116), melanoma (A375), triple-negative breast cancer (MDA-MB-231), and ovarian (IGROV-1). Treatment with 8.5–11.8 μM TrxFTL reduced proliferation of all cancer cells to half in 48 h. This anti-proliferative effect encompasses the p53 pathway since it was significantly reduced in p53-null colon cancer cells (HCT116 p53−/−; GI50 of 25.0 ± 3.0 μM), when compared to the isogenic p53-positive cells (HCT116 p53+/+; GI50 of 8.7 ± 1.8 μM; p < 0.002). This recombinantly produced frutalin isoform has relevant cytotoxic effect and its biological activity is not dependent on glycosylation. The developed E. coli production and purification protocol generates high yield of non-glycosylated frutalin isoform with potent cytotoxic activity, enabling the development of novel anticancer p53-targeting therapies.

New Ultrastructural Observations on Injury and Regeneration of Cilia in Mammalian Conducting Airway Epithelium

1981 ◽  
Vol 39 ◽  
pp. 624-625
Author(s):  
J.L. Carson ◽  
A.M. Collier
Keyword(s):  
Respiratory Tract ◽  
Airway Epithelium ◽  
Defense Mechanisms ◽  
Normal Growth ◽  
Fetal Lung ◽  
Secretory Cells ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Conducting Airway ◽  
Conducting Airways

The ciliated cells lining the conducting airways of mammals are integral to the defense mechanisms of the respiratory tract, functioning in coordination with secretory cells in the removal of inhaled and cellular debris. The effects of various infectious and toxic agents on the structure and function of airway epithelial cell cilia have been studied in our laboratory, both of which have been shown to affect ciliary ultrastructure.These observations have led to questions about ciliary regeneration as well as the possible induction of ciliogenesis in response to cellular injury. Classical models of ciliogenesis in the conducting airway epithelium of the mammalian respiratory tract have been based primarily on observations of the developing fetal lung. These observations provide a plausible explanation for the embryological generation of ciliary beds lining the conducting airways but do little to account for subsequent differentiation of ciliated cells and ciliogenesis during normal growth and development.

Quality films from carbon fiber evaporation for thorough coverage of TEM grids

1988 ◽  
Vol 46 ◽  
pp. 418-419
Author(s):  
N. Tempel ◽  
M. C. Ledbetter
Keyword(s):  
Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Carbon Films ◽  
Low Noise ◽  
Vacuum Evaporation ◽  
High Yield ◽  
Good Adhesion ◽  
Noise Structure ◽  
Resolution Electron ◽  
Carbon Foils

Carbon films have been a support of choice for high resolution electron microscopy since the introduction of vacuum evaporation of carbon. The desirable qualities of carbon films and methods of producing them has been extensively reviewed. It is difficult to get a high yield of grids by many of these methods, especially if virtually all of the windows must be covered with a tightly bonded, quality film of predictable thickness. We report here a method for producing carbon foils designed to maximize these attributes: 1) coverage of virtually all grid windows, 2) freedom from holes, wrinkles or folds, 3) good adhesion between film and grid, 4) uniformity of film and low noise structure, 5) predictability of film thickness, and 6) reproducibility.Our method utilizes vacuum evaporation of carbon from a fiber onto celloidin film and grid bars, adhesion of the film complex to the grid by carbon-carbon contact, and removal of the celloidin by acetone dissolution. Materials must be of high purity, and cleanliness must be rigorously maintained.

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