What is the yeild per liter and for how many days protein is stable in this buffer at 37 degrees? Have you checked its degradation profile over time?

Thanks for your timely response.

Regards

Ishtiyaq

Hi Ishtiyaq,

This depends on the construct you are using. For 297-391 we see yields up to 100mg per litre if expressed in 2xTY with 1 mM IPTG (note, 2xTY is better than TB for this construct, auto-induction using YB or LB is even better). For auto-induction, we pick one colony which is grown for 8 hrs in 1 L and then set the temperature to 24 degrees ON. For induced expression using IPTG, we use all colonies in a plate, let this grow to an OD600 to 0.8 and induce for 3-4 hrs at 37 degrees.

During the purification of the construct, we do not see degradation (nor after filament assembly) - cysteine linked dimers are common which can be removed with some DTT, TCEP etc. Full length tau does degrade during purification if not done at cold temperatures (chymostatin is required during the purfication of full length tau).

Hope this answers your question,

bw,

Sofia