TY - JOUR
T1 - A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins
AU - Hefti, Marco H
AU - Milder, Fin J
AU - Boeren, Sjef
AU - Vervoort, Jacques
AU - van Berkel, Willem J H
PY - 2003/1/20
Y1 - 2003/1/20
N2 - The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution. The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors.
AB - The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution. The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors.
KW - apoproteins/chemistry
KW - azotobacter vinelandii/genetics
KW - bacterial proteins/biosynthesis
KW - chromatography, affinity/methods
KW - circular dichroism
KW - escherichia coli/metabolism
KW - flavin mononucleotide/chemistry
KW - flavin-adenine dinucleotide/chemistry
KW - flavoproteins/biosynthesis
KW - genetic vectors
KW - nuclear magnetic resonance, biomolecular
KW - protein structure, tertiary
KW - recombinant proteins/chemistry
KW - apoproteïnen/chemie
KW - azotobacter vinelandii/genetica
KW - bacteriële eiwitten/biosynthese
KW - chromatografie, affiniteit/methoden
KW - circulair dichroïsme
KW - escherichia coli/metabolisme
KW - flavine-mononucleotide/chemie
KW - flavine-adenine dinucleotide/chemie
KW - flavoproteïnen/biosynthese
KW - genetische vectoren
KW - nucleaire magnetische resonantie, biomoleculair
KW - eiwitstructuur, tertiair
KW - recombinante eiwitten/chemie
U2 - 10.1016/S0304-4165(02)00474-9
DO - 10.1016/S0304-4165(02)00474-9
M3 - Article
C2 - 12527109
SN - 0304-4165
VL - 1619
SP - 139
EP - 143
JO - Biochimica et Biophysica Acta (BBA) - General Subjects
JF - Biochimica et Biophysica Acta (BBA) - General Subjects
IS - 2
ER -