Analysis of three overexpression systems for VanX, the Zinc(II) dipeptidase required for high-level vancomycin resistance in bacteria.
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 approximately 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 microM 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 approximately 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.
Brandt
J J
JJ
Department of Chemistry and Biochemistry, Miami University, 112 Hughes Hall, Oxford, Ohio 45056, USA.
Chatwood
L L
LL
Crowder
M W
MW
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002686:Chitin / Q000378:metabolism; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D016983:Enterococcus / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002686:Chitin; D004150:Dipeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase