Overexpression and Large-Scale Purification of Recombinant Hamster Polymorphic ArylamineN-Acetyltransferase as a Dihydrofolate Reductase Fusion Protein

1997 ◽  
Vol 10 (1) ◽  
pp. 141-153 ◽  
Author(s):  
Kristina R.K. Sticha ◽  
Christine A. Sieg ◽  
Carl P. Bergstrom ◽  
Patrick E. Hanna ◽  
Carston R. Wagner
Keyword(s):  
Fusion Protein ◽  
Dihydrofolate Reductase ◽  
Large Scale

scholarly journals Simvastatin Efficiently Reduces Levels of Alzheimer’s Amyloid Beta in Yeast

2019 ◽  
Vol 20 (14) ◽  
pp. 3531 ◽  
Author(s):  
Sudip Dhakal ◽  
Mishal Subhan ◽  
Joshua M. Fraser ◽  
Kenneth Gardiner ◽  
Ian Macreadie
Keyword(s):  
Fusion Protein ◽  
Amyloid Beta ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Convenient Means ◽  
Ergosterol Synthesis ◽  
Green Fluorescent

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

scholarly journals Accelerated CO2 Hydration with Thermostable Sulfurihydrogenibium azorense Carbonic Anhydrase-Chitin Binding Domain Fusion Protein Immobilised on Chitin Support

2019 ◽  
Vol 20 (6) ◽  
pp. 1494 ◽  
Author(s):  
Juan Hou ◽  
Xingkang Li ◽  
Michal Kaczmarek ◽  
Pengyu Chen ◽  
Kai Li ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Fusion Protein ◽  
Large Scale ◽  
Economic Benefits ◽  
Binding Domain ◽  
Fusion Partner ◽  
Carbonic Anhydrases ◽  
Negative Effects ◽  
Chitin Binding Domain ◽  
Fusion Enzyme

Carbonic anhydrases (CAs) represent a group of enzymes that catalyse important reactions of carbon dioxide hydration and dehydration, a reaction crucial to many biological processes and environmental biotechnology. In this study we successfully constructed a thermostable fusion enzyme composed of the Sulfurihydrogenibium azorense carbonic anhydrase (Saz_CA), the fastest CA discovered to date, and the chitin binding domain (ChBD) of chitinase from Bacillus circulans. Introduction of ChBD to the Saz_CA had no major impact on the effect of ions or inhibitors on the enzymatic activity. The fusion protein exhibited no negative effects up to 60 °C, whilst the fusion partner appears to protect the enzyme from negative effects of magnesium. The prepared biocatalyst appears to be thermally activated at 60 °C and could be partially purified with heat treatment. Immobilisation attempts on different kinds of chitin-based support results have shown that the fusion enzyme preferentially binds to a cheap, untreated chitin with a large crystallinity index over more processed forms of chitin. It suggests significant potential economic benefits for large-scale deployment of immobilised CA technologies such as CO2 utilisation or mineralisation.

Production of Recombinant Melittin by Auto-Induction in Escherichia coli

2013 ◽  
Vol 798-799 ◽  
pp. 1007-1012
Author(s):  
Wen He Zhu ◽  
Wei Zhang ◽  
Yan Li ◽  
Jun Jie Xu ◽  
Shi Jie Lv
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression Vector ◽  
Human Cancer ◽  
Expression System ◽  
Scale Production ◽  
Recognition Sequence ◽  
Large Scale Production ◽  
N Terminus ◽  
Gst Fusion Protein

Melittin is a novel peptide of biological activity isolated from bee venom. It has potential application value in medicine and agriculture. Here we encoded melittin gene with the EK recognition sequence in the N-terminus into expression vector pGEX-2T.The expressed fusion protein, which is about 29KDa, identified by Western Blot. To facilitate large-scale production of recombinant GST-fusion protein, we optimized different expression conditions to increase the overall production of the fusion protein. The production of the protein had increased about 10-fold when we used an auto-inducing medium. The GST fusion protein showed an equivalent activity with the natural melittin after digested by EK and can inhibited the proliferations of several human cancer lines. The expression system described in this study provides a feasible way for producing melittin in further studies.

scholarly journals Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus

2002 ◽  
Vol 83 (3) ◽  
pp. 623-629 ◽  
Author(s):  
Ming Yu ◽  
Enxiu Wang ◽  
Youfang Liu ◽  
Dianjun Cao ◽  
Ningyi Jin ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Large Scale ◽  
Heptad Repeat ◽  
Scale Production ◽  
F Protein ◽  
Helix Bundle ◽  
Fusion Inhibitors

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus–cell fusion inhibition activity (IC50 of 1·07–2·93 μM). The solubility of the GST–HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.

scholarly journals The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

2011 ◽  
Vol 133 (50) ◽  
pp. 20561-20570 ◽  
Author(s):  
E. Joel Loveridge ◽  
Lai-Hock Tey ◽  
Enas M. Behiry ◽  
William M. Dawson ◽  
Rhiannon M. Evans ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Large Scale

scholarly journals Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

2004 ◽  
Vol 70 (6) ◽  
pp. 3292-3297 ◽  
Author(s):  
Gerard M. Gibbs ◽  
Barrie E. Davidson ◽  
Alan J. Hillier
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
Scale Production ◽  
Gene Encoding ◽  
Strong Activity ◽  
E Coli ◽  
Large Scale Production ◽  
Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

scholarly journals Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.

1996 ◽  
Vol 132 (3) ◽  
pp. 311-324 ◽  
Author(s):  
T Häusler ◽  
Y D Stierhof ◽  
E Wirtz ◽  
C Clayton
Keyword(s):  
Fusion Protein ◽  
Trypanosoma Brucei ◽  
Dihydrofolate Reductase ◽  
Fusion Proteins ◽  
Phosphoglycerate Kinase ◽  
Cytochrome Oxidase Subunit ◽  
Conformational Properties ◽  
Terminal Amino ◽  
Carboxy Terminal

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.

scholarly journals Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to diphtheria toxin

1996 ◽  
Vol 313 (2) ◽  
pp. 647-653 ◽  
Author(s):  
Olav KLINGENBERG ◽  
Sjur OLSNES
Keyword(s):  
Fusion Protein ◽  
Dihydrofolate Reductase ◽  
Diphtheria Toxin ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Toxin B ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Diphtheria Toxin A

A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.

scholarly journals Resistance to Methotrexate Due to AcrAB-Dependent Export from Escherichia coli

2000 ◽  
Vol 44 (11) ◽  
pp. 3210-3212 ◽  
Author(s):  
Stephan J. Kopytek ◽  
John C. D. Dyer ◽  
Gwendowlyn S. Knapp ◽  
James C. Hu
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Fusion Protein ◽  
Dihydrofolate Reductase ◽  
Efflux Pump ◽  
In Cells

ABSTRACT Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate eithertolC or acrA confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations inacrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.

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