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@article{842448,
author = {Tesařík, Radek and Sedláček, Vojtěch and Plocková, Jana and Wimmerová, Michaela and Turánek, Jaroslav and Kučera, Igor},
article_number = {2},
keywords = {Quinone reductase; Protein expression in E. coli; His-tag fusion; Light scattering; Cross-linking; FT-IR spectrometry; Differential scanning calorimetry; Thermal inactivation},
language = {eng},
issn = {1046-5928},
journal = {Protein Expression and Purification},
title = {Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans},
volume = {68},
year = {2009}
}
TY - JOUR
ID - 842448
AU - Tesařík, Radek - Sedláček, Vojtěch - Plocková, Jana - Wimmerová, Michaela - Turánek, Jaroslav - Kučera, Igor
PY - 2009
TI - Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans
JF - Protein Expression and Purification
VL - 68
IS - 2
SP - 233-238
EP - 233-238
PB - Elsevier
SN - 10465928
KW - Quinone reductase
KW - Protein expression in E. coli
KW - His-tag fusion
KW - Light scattering
KW - Cross-linking
KW - FT-IR spectrometry
KW - Differential scanning calorimetry
KW - Thermal inactivation
N2 - FerB is a flavoenzyme capable of reducing quinones, ferric complexes and chromate. Its expression in Escherichia coli as a hexahistidine fusion resulted in a functional product only when the tag was placed on the C-terminus. The molecular mass values estimated by gel permeation chromatography were compatible with the existence of either dimer or trimer, whereas the light scattering data, together with cross-linking experiments which yielded exclusively monomer and dimer bands on dodecyl sulfate-polyacrylamide gels, strongly supported a dimeric nature of both native and tagged form of FerB. These two proteins also exhibited almost identical secondary structures as judged by Fourier transform infra red spectrometry. The presence of tag, however, shifted the thermal denaturation curve towards lower temperatures and decreased the thermoresistance of enzyme activity. Despite somewhat lower thermal stability, the fusion protein is considered a better candidate for crystallization than the wild-type one due to a more negative value of its second optical virial coefficient.
ER -
TESAŘÍK, Radek, Vojtěch SEDLÁČEK, Jana PLOCKOVÁ, Michaela WIMMEROVÁ, Jaroslav TURÁNEK a Igor KUČERA. Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. \textit{Protein Expression and Purification}. Elsevier, roč.~68, č.~2, s.~233-238. ISSN~1046-5928. 2009.
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