A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins.
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.
Hefti
Marco H
MH
Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. marco.hefti@wur.nl
Milder
Fin J
FJ
Boeren
Sjef
S
Vervoort
Jacques
J
van Berkel
Willem J H
WJ
D001059:Apoproteins / Q000737:chemistry; D016948:Azotobacter vinelandii / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005420:Flavoproteins / Q000096:biosynthesis / Q000737:chemistry*; D005822:Genetic Vectors; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D001059:Apoproteins; D001426:Bacterial Proteins; D005420:Flavoproteins; D011994:Recombinant Proteins; C084296:nifL protein, Bacteria; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide