Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans
| Authors | |
|---|---|
| Year of publication | 2009 |
| Type | Article in Periodical |
| Magazine / Source | Protein Expression and Purification |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Quinone reductase; Protein expression in E. coli; His-tag fusion; Light scattering; Cross-linking; FT-IR spectrometry; Differential scanning calorimetry; Thermal inactivation |
| Description | 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. |
| Related projects: |