Hi everyone!
We recently had a case where we made some baculoviruses with expression from the polH promoter, and handed over the viruses to the user.  The user reported back to us that he always sees two bands during the purification.  He went so far as to identify these bands via mass spec and discovered that the slightly larger band was his protein with an N-terminal extension resulting from a translation site upstream in the polH promoter.  It is an ATT site, and it is actually described in the Invitrogen/pFastBac manual as a potential cryptic start codon, but is described as a rare event that will not result in large amounts of protein.  In some cases I was told that he sees 50% of the total protein produced with this N-terminal extension.

We have not noticed this with any of our other many constructs, but we do not always purify the proteins ourselves, and it could be that users have simply not reported back to us that they see this double band.  Has anyone else experienced this??

Thanks!

best,

Peggy

Dear Peggy,

Haven't seen something like this with the Bac to Bac system, (these days I'm using mostly BestBac or FlashBac, and another thought: it might be even more elusive when you produce secreted proteins), but my guess is that the initiation codon might not be presented in an optimal way to the ribosome, hence opportunistic initiation occurs. This might happen more often if there is no fusion tag at the N-term of the protein (these are mostely optimized for ribosome binding sites), and when the sequence around the ATG is GC rich. Did the protein express well?

Good luck,

Tsafi

Hi Tsafi!

Thanks for your reply!  I think the protein expressed fairly well, but was not one of our best expressers.  It was cloned with an N-terminal strep tag.  We think we have seen it with one other construct, though we have not confirmed that with mass spec.  That protein also has an N-terminal strep.  My thinking was also that it must have to do with accessibility of the ATG in front of the tag.  We are now debating whether we should modify our vectors to put this ATT site out of frame, but before doing that I wanted to see if anyone else ever observed this.

best,

Peggy

Hi Peggy,

We haven't had these cases ourselves, but a postdoc in a neighbouring lab experiences the same during his PhD. I think they sorted it by mutating the "false" site, but there is a precedent.

See you in December,

Svend