Mesoplasma florum: Tn5 Transposase

Tn5 Transposase production

Tn5 transposase is the key enzyme in forming transposomes for random transposon insertions. It is sold by Epicentre at ridiculously high price. Here, we make it from a plasmid provided by Prof. Wolfgang Hillen PMID 16820464. The protocol also builds on the NEB IMPACT-CN protein purification kit.

Protocol

• Grow BL21(DE3)pLysS cells transformed with pWH1891 in 10 ml culture.
• Inoculate 2 liters of LB/Cm/Amp culture medium with the culture and grow overnight at 23°C
• Induce cultures at OD 0.5 with 50 mM IPTG and grow an additional 5 hours
• Spin down cultures and resuspend in 50 ml TEGX buffer/original liter, transferring to 50 ml centrifuge tubes
• Spin down a second time and resuspend in 10 ml/original liter TEGX + Roche Complete protease inhibitor
• Sonicate 3x pausing 10 minutes between sonications with the cells on ice
• Centrifuge to remove cell debris
• Load a 2 cm diameter column with 20 ml chitin bead suspension (10 ml beads)
• Wash the column with 100 ml TEGX buffer
• Load the cell supernatent onto the column and allow it to flow through
• Flow the supernatent past the column a second time
• Wash the column with 200 ml TEGX buffer
• Add 1 ml of 1M IPTG to 20 ml of TEGX buffer
• Flow the 20 ml IPTG + TEGX into the column
• Cap the column and hold at 4°C overnight
• Recover the protein with 10 ml TEGX buffer flowed through the column