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 14g wet weight cell pellet within 2 days. (C) 2002 Elsevier Science (USA). All rights reserved.