We have cloned a gene into pET20b. The clone was sequenced and the gene is in the correct frame. However, we are having difficulties in expression of the protein. We do not have antibodies to confirm the expression of the protein. Has anyone any experience in such kind of protein expression?
Did you keep the c-term His tag? Perhaps you can overexpress it and then do size exclusion (remember big things come off first). would it bind to pos or neg charged matrix?
To identify it, if you can get something of the right size, I'd say do mass spec.
Thanks Amanda. Yes we have kept the c-term and we have done small scale expression, but the protein is not distinguishable on gel as over-expressed recombinant protein should be.
If you have a His tag you should be able to get an anti-His antibody to show expression via WB.
But yes, sometimes proteins don't express. Your protein seem to be quite small. Maybe your protein is more stable when expressed from pET39 with DsbA tag? I know that doesn't really make sense in terms of disulfides, but you might just want to have something bulky and well folded at the N-term.
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