Hello all,
I am currently trying to modify my plasmids so that it has a sequence coding the pelB or ompA tag in front of the proteins. The problem is that in order to paste such a sequence into the plasmid in the place of interest to me, I have to use the NcoI enzyme sequence at the beginning, which is impossible considering that the pelB or ompA tag must be the first (their sequence begins with Met-Lys- ( ...)), and Lysine have only two codons, none of each is viable. Hence my question, if I add two additional amino acids to the pelB or ompA tag at the beginning so that the result looks like e.g. Met-Gly-Met-Lys- (rest of pelB tag)-(protein), will the pelB tag still work and will it direct the expression to the periplasm, or is its first aminoacids so conservative that it stops working when two amino acids are added?
Thank you for all help,
Szymon
Dear Szymon
i suggest to you to forgot the enzime standard cloning approach and use the PIPE cloning methods.
You have just to design primers to perform your vector PCR (using an high fidelity TAQ) adding to the primers flanking regions which partially overlap able to codify for the signal peptide that you would like to insert or replace.
You can find an example of the primers that i designed some years ago for rhwe insertion of ompA and pelB signal before some genes of our interest.
The only concern regaridg PIPE is that to my knowledge the only commercial strain that work with it are the MACH1 cells.
If you would like to have more information on PIPE cloning, visit my blog ProteoCool (https://proteocool.blogspot.com/) and/or read the attached papers.
good luck
Manuele
Adding N-terminal amino acids to a signal sequence which is at the beginning of a protein might counteract the purpose of the signal sequence. As Manuele suggested, changing the DNA sequence within some constraints might be the better approach, but you need to make sure the promoter(s) still work(s) and the mRNA will be translated (Shine-Delgarno sequence for bacteria / Kozak Consensus for eukaryotes). Extending the sequence by PCR with suitable adapter primers is one way to do it, you don't need a complete NcoI site as long as you can ligate your amplified construct into one after cutting (refer to e.g. the NEB catalogue for compatible cutters). Beware that Taq in PCR reaction digests is happily filling in nucelotides, so don't forget an adequate cleanup before you proceed.
Before things are getting too complicated, consider a (maybe partial) chemical synthesis of the cDNA you're working with, it's cheap nowadays.
I would advise against inserting amino-acids at the N-terminal for two reasons. 1. Might affect the translated protein conformation 2. The stability of the protein thus produced.
However, add your intended residues and try predicting the cleavage with SignalP Server (http://www.cbs.dtu.dk/services/SignalP/) and see if it would still give a clean cleavage. If yes, you're good to go. If not, you might wanna consider alternate cloning strategies. I would recommend using a vector having RE site preceding pelB such as pET-22b (NdeI site).
Good luck!
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