Expression and purification of Rhizobium leguminosarum NodD.
A Rhizobium expression system was constructed via introducing strong transcriptional elements to an IncP broad host range plasmid pKT230. Using this system, the nodD gene of Rhizobium leguminosarum biovar viciae was overexpressed in its own in vivo environment. Western blot showed that the NodD yield of the newly constructed expression vector pKNDT was much higher than that of a reported vector pIJ1518. Based on the result from quantitative gel retardation assay, the specific DNA-binding activity of NodD was estimated to increase by about 80-fold. Purification of NodD was performed by the sequential steps: polyethyleneimine treatment, ammonium sulfate fractional precipitation, and cellulose phosphate P11 chromatography. About 26 mg NodD with a purity about 90% was obtained from 14 g wet weight cell pellet within 2 days.
Feng
Jie
J
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, 200031, Shanghai, PR China.
Qing Li
Feng
F
Li
Qiang
Q
Liang Hu
Hai
H
Fan Hong
Guo
G
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D016961:Rhizobium leguminosarum / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; C060773:NodD protein, Bacteria