I'm trying to express a type I eukaryotic membrane protein (N- terminus inside and C- terminus outside) in E. coli. This protein has one transmembrane domain at C-terminus and a total of 8 disulfide bonds. I expressed it in several strains including Rosetta, Rosetta-gami, and Shuffle T7 (all with pRARE plasmid for rare codons and Lysozyme inhibition). Upon induction with different concentrations of IPTG, I always face with two situation in all strains: some colonies of same strain produce inclusion bodies and some have no corresponding band (60 KDa) at all. Even colonies which produce inclusion bodies may fail for protein expression in different tests while all conditions are kept constant.

I know membrane proteins may be toxic to cells and saturate SRP complex. But actually I did not see any difference in growth of control and recombinant cells. I also collected samples at different hours following IPTG induction to check protein degradation but there was no clear band at any time. Further, I used some solubility tags to help protein expression and membrane integration but they did not worked. I have sequenced my constructs. Everything is fine. Deletion of N-and C- terminal hydrophobic parts resulted in inclusion bodies that cannot be refolded.

I need this protein to be integrated in bacterial membrane, I would be very grateful if someone help me to answer why some colonies cannot express the protein while others produce it as inclusion bodies.