I am expressing a scfv antibody (fusion protein) and since most scfv expressed are insoluble, i decided to use the plasmid pet 26b which has a pelb sequence for periplasmic translocation. The host is bl21 de3 plyss.
However, I couldnt use the his tag at the C terminal (to not interfer with a peptide fused to my scfv at the C ter), so I put the his tag at the N terminal.
The his tag is after pelb sequence because i dont want it to be removed when the fp protein reaches periplamic space, pelb sequence is supposed to be removed. I knew that his-tag (n ter) could interfer with pelb sequence but some people have done this before. Actually they got extracellular secretion, because his-tag in N terminal enhance extracellular secretion when there is a pelb sequence at N-ter as well.
Unfortunately, my protein is expressing in inclusion bodies (sds page after extraction), i guess his-tag is interfering with pelb signal sequence. I would like to know if someone has done this, denaturing the inclusion bodies and then refolding. Have I a good chance to get the correct re folding ?
What can I do else ? What I have read about denaturing refolding is : time consuming, expensive etc.... Is there a easy and cheap protocol for refolding purification ?
Thanks very much !