pET32b+ vector issue - (Mar/23/2016 )
Hello fellow Scientists,
I'm currently working with the expression vector pET32b+ (Novagen). I have been trying the insert a DNA sequence into the vector in sillco, without success. After trying once and again, I downloaded the vector manual and saw that ver a, b and c differ in the MCS, as shown below.
http://www.helmholtz-muenchen.de/fileadmin/PEPF/pET_vectors/pET-32a-c_map.pdf
As shown in the manual, ver b has one less letter in the MCS. This in turn is changing the ORF entirely and is causing my insert not to have the His-Tag at the end.
I'm using RE Nco I and Sal I.
Do I take the reading frame as lined up against a ?? or do I take the Nco I site as indicated in the manual (CCATGGC)?? The normal RE Nco I is CCATGG
Can someone help?
Hello delf0s,
1. Reading frames start at the ATG. Importantly, NcoI contains ATG, and your sequence must be in frame with that ATG to be properly expressed. The presence of any changes downstream of that start site will only effect the resulting sequence/stop sites, but the reading frame will be the same. I can't tell based on your initial post, but if you have an independent start site in your sequence, it may not be highly expressed as the initial ATG--> AUG is likely to be favored for translation. I would presume that if you're using NcoI, you are intending to start translation from that start codon.
2. I quickly looked over the link you attached. The difference I see between the a/b/c vectors is not in the NcoI open reading frame itself (i.e. the start site ATG is not different if you clone into NcoI properly). Instead, the sequence of the ORF changes beyond the NcoI site. In this case a, b, and c encode different linkers and "b" encodes a c-terminal 6x poly HIs-tag if kept in frame with your insert.
Hence, if you are sub-cloning your insert from one vector to another with RE digest/ligation, you will run into a problem if the sequence downstream of the second RE site (SalI in your case) is out of frame with your insert. So if you find the His-tag out of frame, it has to do with SalI being shifted with respect to the NcoI site (which is CCATGG as you've indicated).
If you want to use NcoI and SalI, I'd suggest using a PCR cloning method. Alternatively, perhaps one of the other restriction sites will work for you and keep the 6x-His in-frame?