Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 327548
Title A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins
Author(s) Hefti, M.H.; Milder, F.J.; Boeren, J.A.; Vervoort, J.J.M.; Berkel, W.J.H. van
Source Biochimica et Biophysica Acta. General subjects 1619 (2003)2. - ISSN 0304-4165 - p. 139 - 143.
DOI https://doi.org/10.1016/S0304-4165(02)00474-9
Department(s) Biochemistry
EPS-1
Publication type Refereed Article in a scientific journal
Publication year 2003
Keyword(s) para-hydroxybenzoate hydroxylase - pseudomonas-fluorescens - azotobacter-vinelandii - pas domain - flavin - riboflavin - protein - oxidase - nifl - chromatography
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-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.
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