A His-tag based immobilization method for the preparation and
reconstitution of apoflavoproteins
Marco H. Hefti
, Fin J. Milder, Sjef Boeren, Jacques Vervoort, Willem J.H. van Berkel
Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenlaan 3,
6703 HA Wageningen, The Netherlands
Received 18 July 2002; received in revised form 1 October 2002; accepted 16 October 2002
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-
C-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.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Apoflavoprotein; Deflavination; Flavin; His-tagged protein; IMAC; Immobilization; PAS domain; Reconstitution; NifL
The reversible dissociation of flavoproteins into its con-
stituents, apoprotein and flavin prosthetic group, has received
much attention [1–3]. Generation of a stable soluble form of
the apoprotein is of great interest as it allows the reconstitu-
tion of the holoprotein with either artificial [4–6], enzymati-
cally modified  or isotopically enriched flavin analogues
[8–10]. Conventional methods of apoprotein preparation
include acid ammonium sulfate precipitation , dialysis
in the presence of chaotropic salts  or treatment with
unfolding agents [13,14]. These methods can lead to signifi-
cant irreversible protein denaturation and are often not suited
for large-scale applications. Therefore, we introduced the
concept of reversible protein immobilization for improving
the yield and reconstitutability of the apoprotein .
For p-hydroxybenzoate hydroxylase (PHBH) from Pseu-
domonas fluorescens, it was shown that large amounts of
highly reconstitutable apoprotein can be prepared by cova-
lently binding the enzyme to thiol agarose . The suit-
ability of this mild procedure was confirmed by the high-
resolution crystal structure of PHBH reconstituted with
arabino-FAD . However, the thiol affinity chromatogra-
phy method requires the presence of a freely available thiol
group at the protein surface, making the method not gen-
erally applicable . Therefore, another immobilization
procedure was developed for the large-scale preparation of
apo flavoproteins using hydrophobic interaction chromatog-
raphy (HIC) .
This method makes use of the fact that many flavoproteins
bind to phenyl agarose at neutral pH in the presence of 1 M
ammonium sulfate. After immobilization, the flavin can be
released by the addition of a high concentration of KBr and/
or lowering the pH of the elution buffer. The HIC method has
been successfully applied for a number of flavoproteins [16 –
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
Abbreviations: HIC, hydrophobic interaction chromatography; IMAC,
immobilized metal-ion affinity chromatography; NifL, nitrogen fixation re-
gulatory protein L; Ni-NTA, nickel-nitrilotriacetic acid; PAS domains, PER-
ARNT-SIM homology regions; PHBH, p-hydroxybenzoate hydroxylase
* Corresponding author. Tel.: +31-317-482-868; fax: +31-317-484-801.
E-mail address: email@example.com (M.H. Hefti).
Biochimica et Biophysica Acta 1619 (2003) 139–143