Enzymatic Characterization of Recombinant Human Dihydrofolate Reductase Produced in E. coli

1986 ◽  
pp. 839-842 ◽  
Keyword(s):  
Dihydrofolate Reductase ◽  
Enzymatic Characterization ◽  
E Coli ◽  
Human Dihydrofolate Reductase

scholarly journals Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

2017 ◽  
Vol 8 (5) ◽  
pp. 4062-4072 ◽  
Author(s):  
Michael Cammarata ◽  
Ross Thyer ◽  
Michael Lombardo ◽  
Amy Anderson ◽  
Dennis Wright ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Dihydrofolate Reductase ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
E Coli ◽  
Exclusion Chromatography

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

scholarly journals Cloning, Expression, and Enzymatic Characterization of Pseudomonas aeruginosaTopoisomerase IV

1999 ◽  
Vol 43 (3) ◽  
pp. 530-536 ◽  
Author(s):  
Takaaki Akasaka ◽  
Yoshikuni Onodera ◽  
Mayumi Tanaka ◽  
Kenichi Sato
Keyword(s):  
Dna Gyrase ◽  
Fusion System ◽  
Enzymatic Characterization ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Topoisomerase Iv ◽  
Upstream Gene ◽  
E Coli ◽  
New Quinolones

ABSTRACT The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified asP. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities ofP. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-typeP. aeruginosa.

scholarly journals Structural Insights of Three 2,4-Disubstituted Dihydropyrimidine-5-carbonitriles as Potential Dihydrofolate Reductase Inhibitors

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3286
Author(s):  
Lamya H. Al-Wahaibi ◽  
Althaf Shaik ◽  
Mohammed A. Elmorsy ◽  
Mohammed S. M. Abdelbaky ◽  
Santiago Garcia-Granda ◽  
...  
Keyword(s):  
Water Molecule ◽  
Dihydrofolate Reductase ◽  
Crystal Packing ◽  
Docking Simulation ◽  
X Ray Diffraction ◽  
Space Groups ◽  
Reductase Inhibitors ◽  
Inhibitory Potential ◽  
Human Dihydrofolate Reductase

In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile 1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 2, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate 3. An X-ray diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water molecule. A cyclic motif (R22(8)) mediated by N–H···O hydrogen bond was formed in compounds 1 and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3. The crystal packing of these compounds was analyzed based on energy frameworks performed at the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate reductase (DHFR) enzyme.

Overexpression of human antiquitin in E. coli: Enzymatic characterization of twelve ALDH7A1 missense mutations associated with pyridoxine-dependent epilepsy

2012 ◽  
Vol 106 (4) ◽  
pp. 478-481 ◽  
Author(s):  
Marion B. Coulter-Mackie ◽  
Ailin Li ◽  
Qun Lian ◽  
Eduard Struys ◽  
Sylvia Stockler ◽  
...  
Keyword(s):  
Missense Mutations ◽  
Enzymatic Characterization ◽  
E Coli

Enzymatic Characterization of Lactococcus lactis subsp. lactis Cyclomaltodextrinase Expressed in E. coli

2013 ◽  
Vol 41 (4) ◽  
pp. 391-397 ◽  
Author(s):  
Myoung-Uoon Jang ◽  
Hye-Jeong Kang ◽  
Chang-Ku Jeong ◽  
Jung-Mi Park ◽  
Ah-Rum Yi ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Enzymatic Characterization ◽  
E Coli ◽  
Lactococcus Lactis Subsp

Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase

2016 ◽  
Vol 128 ◽  
pp. 81-85 ◽  
Author(s):  
Romy Perez-Abraham ◽  
Karla Garabiles Sanchez ◽  
Melany Alfonso ◽  
Ueli Gubler ◽  
John J. Siekierka ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Brugia Malayi ◽  
Enzymatic Characterization

High-resolution gold labeling

1993 ◽  
Vol 51 ◽  
pp. 330-331
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya ◽  
Kyra Carbone ◽  
Martha Simon ◽  
Beth Lin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Polypeptide Chain ◽  
External Surface ◽  
Gold Cluster ◽  
Macromolecular Complex ◽  
Gold Compound ◽  
Central Cavity ◽  
Chain Folding ◽  
E Coli ◽  
Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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