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

doi:10.1016/S0304-4165(02)00474-9

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

Wageningen University & Research

Research output: Contribution to journal › Article › Academic › peer-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 language	English
Pages (from-to)	139-43
Number of pages	5
Journal	Biochimica et Biophysica Acta (BBA) - General Subjects
Volume	1619
Issue number	2
DOIs	https://doi.org/10.1016/S0304-4165(02)00474-9
Publication status	Published - 20 Jan 2003
Externally published	Yes

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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10.1016/S0304-4165(02)00474-9

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title = "A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins",

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.",

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, apoprote{\"i}nen/chemie, azotobacter vinelandii/genetica, bacteri{\"e}le eiwitten/biosynthese, chromatografie, affiniteit/methoden, circulair dichro{\"i}sme, escherichia coli/metabolisme, flavine-mononucleotide/chemie, flavine-adenine dinucleotide/chemie, flavoprote{\"i}nen/biosynthese, genetische vectoren, nucleaire magnetische resonantie, biomoleculair, eiwitstructuur, tertiair, recombinante eiwitten/chemie",

author = "Hefti, \{Marco H\} and Milder, \{Fin J\} and Sjef Boeren and Jacques Vervoort and \{van Berkel\}, \{Willem J H\}",

year = "2003",

month = jan,

day = "20",

doi = "10.1016/S0304-4165(02)00474-9",

language = "English",

volume = "1619",

pages = "139--43",

journal = "Biochimica et Biophysica Acta (BBA) - General Subjects",

issn = "0304-4165",

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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 -

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

Abstract

Keywords

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