scholarly journals An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization

2013 ◽  
Vol 88 (1) ◽  
pp. 80-84 ◽  
Author(s):  
Livia Regina Manzine ◽  
Alexandre Cassago ◽  
Marco Túlio Alves da Silva ◽  
Otavio Henrique Thiemann
Keyword(s):  
Escherichia Coli ◽  
Biophysical Characterization

scholarly journals Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli

2016 ◽  
Vol 291 (11) ◽  
pp. 5803-5816 ◽  
Author(s):  
Robin L. Cassady-Cain ◽  
Elizabeth A. Blackburn ◽  
Husam Alsarraf ◽  
Emil Dedic ◽  
Andrew G. Bease ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factor ◽  
Biophysical Characterization

scholarly journals Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

2000 ◽  
Vol 182 (12) ◽  
pp. 3544-3552 ◽  
Author(s):  
Kristin C. Boesch ◽  
Ruth E. Silversmith ◽  
Robert B. Bourret
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Random Mutagenesis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Functional Impairments ◽  
Biophysical Characterization ◽  
Mutant Proteins ◽  
Isolation And Characterization

ABSTRACT The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ1–189 peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2021 ◽  
Vol 271 ◽  
pp. 106554
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M.N. Scapin ◽  
William de Andrade ◽  
Maíra Fasciotti ◽  
Mariana T.Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biophysical Characterization

scholarly journals Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic β-oxidation trifunctional enzyme complexes

2019 ◽  
Vol 476 (13) ◽  
pp. 1975-1994 ◽  
Author(s):  
Shiv K. Sah-Teli ◽  
Mikko J. Hynönen ◽  
Werner Schmitz ◽  
James A. Geraets ◽  
Jani Seitsonen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Studies ◽  
Biophysical Characterization ◽  
Aerobic Conditions ◽  
Membrane Bound ◽  
Quaternary Assembly ◽  
Enzyme Complexes ◽  
Aerobic And Anaerobic ◽  
Long Chain

AbstractThe trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

scholarly journals Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem

1996 ◽  
Vol 315 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Wendy A. DOYLE ◽  
Andrew T. SMITH
Keyword(s):  
Escherichia Coli ◽  
Phanerochaete Chrysosporium ◽  
Specific Activity ◽  
Spectral Characteristics ◽  
Protein A ◽  
Veratryl Alcohol ◽  
Active Enzyme ◽  
Native Enzyme ◽  
Biophysical Characterization

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and pro-sequence regions of Lip isoenzyme H8 (Lip) has been successfully overexpressed in Escherichia coli. The recombinant protein (LipP*) was sequestered in inclusion bodies. The reduced–denatured polypeptide has been purified by differential solubilization, and the active enzyme recovered after controlled in vitro refolding (albeit in low yield), by glutathione-mediated oxidation of disulphides, in a folding medium containing an intermediate concentration of urea, Ca2+ and haem. The procedure is analogous to that previously described for the production of active recombinant horseradish peroxidase (HRP-C*) from inclusion-body material. It is quite possible, therefore, that this type of procedure may be suitable for the recovery of most, if not all, active recombinant peroxidases. The resultant LipP* has spectral characteristics identical with that of the native enzyme as isolated from Phanerochaete chrysosporium. Its specific activity measured in the standard veratryl alcohol (VA) assay was 39 μmol of VA oxidized/min per mg of protein, a value which compares extremely favourably with that of the native enzyme (36 μmol of VA/min per mg). Although levels of active enzyme obtained are not yet as high as in the case of HRP-C* (1% conversion of crude inactive LipP* polypeptide into pure fully active Lip), it is envisaged that further refinement of the expression/folding/activation procedures will provide sufficient protein for biophysical characterization of both the wild-type and site-directed mutants.

Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli

2010 ◽  
Vol 1804 (9) ◽  
pp. 1902-1907 ◽  
Author(s):  
Tharangani Rathnayaka ◽  
Minako Tawa ◽  
Shihori Sohya ◽  
Masafumi Yohda ◽  
Yutaka Kuroda
Keyword(s):  
Escherichia Coli ◽  
Gaussia Luciferase ◽  
Biophysical Characterization ◽  
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