TY - JOUR
T1 - Expression and Characterisation of Recombinant Rhodocyclus tenuis High Potential Iron-Sulphur Protein
AU - Caspersen, Michael Bjerg
AU - Bennet, K.
AU - Christensen, Hans Erik Mølager
PY - 2000
Y1 - 2000
N2 - The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2.11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe4S4] cluster in the holoprotein, The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein, The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster. (C) 2000 Academic Press.
AB - The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2.11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe4S4] cluster in the holoprotein, The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein, The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster. (C) 2000 Academic Press.
U2 - 10.1006/prep.2000.1242
DO - 10.1006/prep.2000.1242
M3 - Journal article
VL - 19
SP - 259
EP - 264
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 2
ER -