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Construction of a Chimeric ArsA-ArsB Protein for Overexpression of the Oxyanion-translocating ATPase*

Dou, Dexian and Owolabi, Joshua B. and Dey, Saibal and Rosen, Barry P. (1992) Construction of a Chimeric ArsA-ArsB Protein for Overexpression of the Oxyanion-translocating ATPase*. The Journal of Biological Chemistry, 267 (36). pp. 25768-25775.

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Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodesa n ATP-driven anion extrusion pump. The ars operon is composed of three structuralg enes, arsA, arsBa, nd arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amounot f ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integrmale mbrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress tahres B gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (AG = -17 kcal/mol) between the third and twelftcho dons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3’ in frame to the first 1458b ase pairs of the arsA gene to encodea 914-residue chimeric protein (486 residouf eths e ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiatinmg ethionine. The ArsA-ArsB chimera has been overexpressed at -15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO; from cells, demonstrating that the chimera can function as a component of the oxyaniontranslocating ATPase.

Item Type: Article
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Medicine, Health and Life Sciences > School of Biological Sciences
Depositing User: Mrs Patricia Nwokealisi
Date Deposited: 20 Oct 2014 12:01
Last Modified: 20 Oct 2014 12:01

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