Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistan ce plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic A s(LII) (arsenite). In order to study the structure of ArsC and to unravel b iochemical and physical properties of this redox enzyme, wild type enzyme a nd a number of cysteine mutants were overproduced soluble in Escherichia co li. In this paper we describe a novel purification method to obtain high pr oduction levels of highly pure enzyme. A reversed-phase method was develope d to separate and analyze the many different forms of ArsC. The oxidation s tate and the methionine oxidized forms were determined by mass spectroscopy . (C) 2000 Elsevier Science B.V. All rights reserved.