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-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.
Messens
J
J
Dienst Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, St. Genesius-Rode, Belgium. jmessens@vub.ac.be
Hayburn
G
G
Brosens
E
E
Laus
G
G
Wyns
L
L
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D053501:Arsenite Transporting ATPases; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016623:Ion Pumps; D013058:Mass Spectrometry; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology*
D016623:Ion Pumps; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases