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

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• 作者：Hefti ; Marco H. ; Milder ; Fin J. ; Boeren ; Sjef ; Vervoort ; Jacques ; van Berkel ; Willem J.H.
• 关键词：Apoflavoprotein ; Deflavination ; Flavin ; His-tagged protein ; IMAC ; Immobilization ; PAS domain ; Reconstitution ; NifL ; HIC ; hydrophobic interaction chromatography ; IMAC ; immobilized metal-ion affinity chromatography ; NifL ; nitrogen fixation regulatory protei
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2003
• 出版时间：January 20, 2003
• 年：2003
• 卷：1619
• 期：2
• 页码：139-143
• 全文大小：172 K

文摘

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.