PMID:11049753
| Citation |
Brandt, JJ, Chatwood, LL and Crowder, MW (2000) Analysis of three overexpression systems for VanX, the Zinc(II) dipeptidase required for high-level vancomycin resistance in bacteria. Protein Expr. Purif. 20:300-7 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/prep.2000.1303 |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Chitin/metabolism; Chromatography, Affinity; Circular Dichroism; Cloning, Molecular; Dipeptidases/chemistry; Dipeptidases/genetics; Dipeptidases/isolation & purification; Dipeptidases/metabolism; Drug Resistance, Microbial/genetics; Electrophoresis, Polyacrylamide Gel; Enterococcus/enzymology; Enterococcus/genetics; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Vectors/genetics; Kinetics; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Serine-Type D-Ala-D-Ala Carboxypeptidase; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
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