Overproduction and Purification of the Tn3 Transposase

抄録

The Tn3 transposase accumulated to about 4% of total cell protein in a minicellproducing Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: i) derepression of transcription resulting from inactivation of the repressor gene (tnpR); ii) efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, iii) the dosage effect of the increased plasmid copy number resulting from deletion of the rom gene of the plasmid; and iv) use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; i) sonication, ii) precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, iii) ammonium sulfate precipitation, iv) gel filtration through Sephacryl S-300, and v) 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.