Identification of a Novel Vanadium-binding Protein by EST Analysis on the Most Vanadium-rich Ascidian, Ascidia gemmata
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Ascidians are known to accumulate extremely high levels of vanadium in their blood cells (up to 350 mM). The branchial sac and the intestine are thought to be the first tissues to contact the outer environment and absorb vanadium ions. The concentration of vanadium in the branchial sac and the intestine of the most vanadium-rich ascidian A. gemmata were determined to be 32.4 mM and 11.9 mM, respectively. Using an expressed sequence tag (EST) analysis of a cDNA library from the intestine of A. gemmata, we determined 960 ESTs and found 55 clones of metal-related gene orthologs, 6 redox-related orthologs, and 18 membrane transporter orthologs. Among them, 2 genes, exhibited significant similarity to the vanadium-binding proteins of other vanadium-rich ascidian species, were designated AgVanabin1 and AgVanabin2. Immobilized metal ion affinity chromatography revealed that recombinant AgVanabin1 bound to metal ions with an increasing affinity for Cu(II) > Zn(II) > Co(II), and AgVanabin2 bound to metal ions with an increasing affinity for Cu(II) > Fe(III) > V(IV). To examine the use of AgVanabins for a metal absorption system, we constructed Escherichia coli strains that expressed AgVanabin1 or AgVanabin2 fused to maltose binding protein and secreted into the periplasmic space. We found that the strain expressing AgVanabin2 accumulated about 13.5 times more Cu(II) ions than the control TB1 strain. Significant accumulation of vanadium was also observed in the AgVanabin2-expressing strain as seen by a 1.5-fold increase.
Financial support was provided in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#20570070 and #21570077).
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Copyright (c) Springer Science+Business Media, LLC 2011
The final publication is available at Springer via http://dx.doi.org/10.1007/s10126-011-9396-1.
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Graduate School of Science