Tabtoxin-resistant protein: overexpression, purification, and characterization

Protein Expr Purif. 2002 Apr;24(3):439-44. doi: 10.1006/prep.2001.1586.

Abstract

One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyltransferases / genetics*
  • Acetyltransferases / isolation & purification
  • Acetyltransferases / metabolism
  • Azetidines / pharmacology*
  • Bacterial Proteins*
  • Circular Dichroism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Inactivation, Metabolic
  • Isoelectric Point
  • Molecular Weight
  • Pseudomonas / genetics*
  • Spectrometry, Fluorescence

Substances

  • Azetidines
  • Bacterial Proteins
  • tabtoxinine beta-lactam
  • Acetyltransferases
  • tabtoxin-resistance protein, Pseudomonas syringae