tau/pET29b Notes

Protocol for Expression

The following note was submitted by Dr Julien Lefèvre.

I performed over-expression of Tau with the plasmid tau/pET29b. This plasmid works very well. I used this protocol for the over-expression and purification:

E. coli BL21 DE3 Gold competent cells were transformed with the h-Tau40 pET29b plasmid (catalog # 16316, Addgene, Cambridge, MA, USA). Bacteria were grown in LB in the presence of kanamycin (100 ug/mL). Overexpression was realized in presence of 1 mM IPTG during 3,5 hours at 37°C. Cells were boiled during 20 minutes in MES KOH pH 6,9 50 mM, NaCl 500 mM, MgCl2 1 mM and DTT 5 mM. Lysate was then ultracentrifuged at 100000 g during 45 minutes and the protein was diluted in 20 mM MES KOH pH=6,9, 5 mM NaCl, 1 mM DTT. The sample was hydrolyzed by acid (4M HCl) and HPLC for quantification. The concentration of our different productions was calibrated after this treatment. The final yield is quite good.