Tabtoxin-resistant protein: overexpression, purification, and characterization.
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.
Liu
Jinyuan
J
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P. R. China. liujy@mail.tsinghua.edu.cn
Le
Yi
Y
Ye
Bin
B
Zhen
Yun
Y
Zhu
Chunming
C
Shen
Jian
J
Zhang
Riqing
R
D000123:Acetyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001384:Azetidines / Q000494:pharmacology*; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008658:Inactivation, Metabolic; D007526:Isoelectric Point; D008970:Molecular Weight; D011549:Pseudomonas / Q000235:genetics*; D013050:Spectrometry, Fluorescence
D001384:Azetidines; D001426:Bacterial Proteins; C051368:tabtoxinine beta-lactam; D000123:Acetyltransferases; C441636:tabtoxin-resistance protein, Pseudomonas syringae