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

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)139-43
Number of pages5
JournalBiochimica et biophysica acta
Volume1619
Issue number2
DOIs
Publication statusPublished - 20 Jan 2003
Externally publishedYes

Keywords

  • apoproteins/chemistry
  • azotobacter vinelandii/genetics
  • bacterial proteins/biosynthesis
  • chromatography, affinity/methods
  • circular dichroism
  • escherichia coli/metabolism
  • flavin mononucleotide/chemistry
  • flavin-adenine dinucleotide/chemistry
  • flavoproteins/biosynthesis
  • genetic vectors
  • nuclear magnetic resonance, biomolecular
  • protein structure, tertiary
  • recombinant proteins/chemistry

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