Overproduction and purification of the Tn3 transposase.
The Tn3 transposase accumulated to about 4% of total cell protein in a minicell-producing Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: derepression of transcription resulting from inactivation of the repressor gene (tnpR); efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, the dosage effect of the increased plasmid copy number resulting from deletion of the rom gene of the plasmid; and use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; sonication, precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, ammonium sulfate precipitation, gel filtration through Sephacryl S-300, and phosphocellulose and DNA-cellulose chromatography. Milligram quantities of pure transposase can be obtained from one gram of wet cells. A small fraction of the accumulated transposase had a blocked amino-terminus and was eluted separately from the normal protein in the chromatography.
Morita
M
M
Laboratory of Biochemistry, Kansai Medical University, Osaka.
Tsunasawa
S
S
Sugino
Y
Y
D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D019895:Transposases
D009713:Nucleotidyltransferases; D019895:Transposases