The gene from Enterococcus faecilis encoding the dipeptidase VanX was subcloned into overexpression vectors pET-5b, pET-27b, and IMPACT-T7, and VanX was overexpressed in BL21(DE3) pLysS Escherichia coli. The pET-5b-vanx overexpression plasmid produces VanX at similar to 12 mg/L under optimum conditions. VanX produced from this overexpression system exists primarily as a dimer in solution, binds ca. 1 Zn(II) ion per monomer, and exhibits K-m and k(cat) values of 500 +/- 40 muM and 0.074 +/- 0.001 s(-1), respectively, when L-alanine-p-nitroanilide is used as substrate. The IMPACT-T7-vanx overexpression plasmid produces a VanX-fusion protein with a chitin-binding domain that allows for purification of the fusion construct with a chitin column. Cleavage of the fusion protein is completed by an on-column chemical cleavage, resulting in similar to 10 mg/L of purified VanX. VanX produced from this system is identical to that produced from the pET-5b system, except the CD spectrum of the IMPACT-T7-produced VanX suggests a small change in secondary structure. This change in secondary structure does not affect any of the kinetic or metal-binding properties of the enzyme. The pET-27b-vanx overexpression plasmid produces and secretes VanX into the growth medium; this system allows for 20 mg of VanX to be isolated per liter of growth medium. The pET-27b-produced VanX is identical to that produced from pET-5b.