Protein expression

Hi Elizabeth - 

Expressing the TAD was certainly a challenge. There were several key steps we needed to take in order to express large quantities of TAD. 

1. We found the minimal required binding regions for the respective partners by ITC (smaller constructs were less sensitive to degradation). This was helpful primarily for our structural studies, less so for our mechanistic studies. 

2. We only induced for 4 hours. This was critical as we saw that the protein would break down in the cell as a function of time. In addition, after induction we would drop the temperature to 25 C for the remainder of the induction. 

3. Not in this study in particular, but in general I have seen some folks have better TAD success with codon optimization.

4. Our lysis buffer had EDTA, protease inhibitor cocktail, and PMSF. All reagents were ice cold and we worked very fast after harvest. 

I hope this was helpful and do let me/us know if we can help with more specific protocols that are absent from the paper - these general guidelines proved helpful for us. 

All the best, 

Caileen Brison, PhD

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