I am using pET24a and I would like to use the NheI and XhoI sites to add our gene. If I use the NheI site at the N-terminal, the first methionine will be from the vector itself, and not the gene (I can't use the NdeI site that would incorporate the starting Met because it is also located in the middle of our gene). The N-terminal side of this construct is very sequence dependent so I would like to keep only the orignial amino acids. Should I use the full N-terminal sequence of our gene and mutate the starting Met in the vector to some other residue? This would push the starting Met 3 codons further away from the rbs. I could also design the primer to use the vector's methionine (3 amino acids shorter) but then I would have to do mutagenesis on the two codons after the start site to get the original sequence back. I guess my question can be summarized as: will moving the starting Met three codons away from the rbs have an effect on the over expression of the protein in E. coli? Is the positioning of the first Met optimized in pET vectors?
Hi there,
In pET28a plasmid the initiation codon is actually in the NcoI site which is far upstream from bth NdeI and NheI. The 2 different ATG codons you are talking about are in frame with the one from the NcoI site which means translation would start from the NcoI site unless you mutate it. If you do so the distance between RBS and ATG would be too large (around 70 bases) for an efficient initiation.
Dominique, Thanks for catching that. I was originally going to use pET28a but the NcoI site was not compatible with my construct. I must have had it on the brain still. I am using pET24a.
Hi again,
Of course the distance between RBS and initiation codon is optimal in pET vectors. Therefore if you increase the distance between the 2 you expect decrease in translation efficiency. If I were you I would first try the NheI XhoI cloning without modifying the initiation site. The product will contain 3 extra aas at Nter (Met Ala Ser) which shouldn't provoke any dramatic effect. And consider alternative cloning approach only if something wrong results from the presence of these extra aas...
Thanks, Dominique. This part of the structure is a mitochondrial import sequence so I really don't want to add amino acids to it. I will design a primer to start 3 amino acids into our protein, then mutate the 2 amino acids after the start Met so that it matches the native sequence.
Take into account that even if you manage to put the N-terminal sequence of your protein straight after the vector's ATG, there is still no guarantee that you will get the native N-terminal, as removal of the N-terminal methionine by MAP depends on the bulkiness of the second aminoacid (See e.g. PMID 20521764).
I think Dominique's suggestion makes the most sense -try the construction with the 3 extra N-terminal residues first and if that doesn't work, then you can try alternatives such as SUMO fusions, etc.
Excellent suggestions so far-the RBS to ATG should be between 7 and 17 bases so from pET24 you would only have initiation of translation from the NcoI site.
I would consider either mutating out your internal NcoI site in your 'donor' vector using quikchange, re-sequence to confirm and then sub-clone your gene by adding a new 5' NcoI site by PCR. Unfortunately you will have to re-sequence the whole gene after cloning.
Alternatively you could amplify the gene out by PCR, again adding NcoI site at 5' end but then sub-clone using InFusion/EZclone etc. that does not require digestion of the PCR product. Unfortunately you will have to re-sequence the whole gene after cloning.
Depending on your second codon (if it starts with a G) the first method could give you a native N-terminus. The InFusion-based methods will give you your native N-terminus regardless of the second codon as the NcoI 'overhang' is only CATG-you have free reign with the next bases.
You didn't say if you wanted to keep an affinity tag-if you want a near native n-term and c-term His-tag you could also use something like pET 28 or PET21a (NheI-XhoI if you are lucky with reading frames!).
SUMO is also an excellent choice but from most vectors you will still add extra a.a.s in the cloning process after the ...QIGG protease cleavage site. The pOPINS vector and InFusion would again give you a native N-term after SUMO protease cleavage and could dramatically increase your yield of folded protein. This plasmid has His-tag for purification that is removed along with SUMO after protease treatment.
You can try Gibson assembly cloning, it provides full flexibility and independent on restriction sites
https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit
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