Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.

Balogh, Ria Katalin and Gyurcsik, Béla and Hunyadi-Gulyás Éva, Csilla and Christensen, Hans E. M. and Jancsó, Attila (2016) Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein. PROTEIN EXPRESSION AND PURIFICATION, 123. pp. 90-96. ISSN 1046-5928

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Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure.

Item Type: Article
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Depositing User: MTMT SWORD
Date Deposited: 22 Feb 2017 09:18
Last Modified: 22 Feb 2017 09:18

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