u/CompetitionOk1383

I am trying to induce expression of two recombinant proteins in BL21 E. coli cells using Tet induction system. I grew the bacteria to OD 0.5-0.6, induced with 200ng/ml aTc at 16 degrees overnight and purify using Strep-Tag II afinity based column.

I have two issues:
a) For protein A, there is in vivo clevage of recombinant protein.

b) For protein B, protein is majorly in the insoluble fraction after clarification.

I have done some troubleshooting experiments, trying to change the tempertaure, inoculum density, duration of induction, but nothing is changed.

What can i try next??