I have done cloning with PPR virus in 33b, I have confirmed it with Colony PCR, R.E. Digestion and even sequencing I have cloned into BL21(PlysS) and got expressed. My expected protein size is 45 and I got the same too. Since I was hospitalised I couldn continue so I have stored the pellet at -80 After a month I started the work and I couldn get the protein so I revived from the stab and even Glycerol stock even still no bands of my expected size. Actually I have lysed with 2x cell lysis from sigma and even bacterial lysis which I made. I have found that my protein is in insoluble form. I have attached my expressed SDS gel pictures too. Too tensed I was about to purify and check the reactivity ELISA now couldn do anything.
Someone please find a solution what could be wrong?