A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins

M.H. Hefti, F.J. Milder, J.A. Boeren, J.J.M. Vervoort, W.J.H. van Berkel

Research output: Contribution to journalArticleAcademicpeer-review

35 Citations (Scopus)


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-13-FAD or 2,4a-C-13-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. (C) 2002 Elsevier Science B.V. All rights reserved.
Original languageEnglish
Pages (from-to)139-143
JournalBiochimica et Biophysica Acta. General subjects
Issue number2
Publication statusPublished - 2003


  • para-hydroxybenzoate hydroxylase
  • pseudomonas-fluorescens
  • azotobacter-vinelandii
  • pas domain
  • flavin
  • riboflavin
  • protein
  • oxidase
  • nifl
  • chromatography


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