Cloning of 9mer nucleotide (3 amino acid) sequence! - (Aug/28/2014 )
Hello everyone, I would like to clone 9 mer nucleotide (3 amino acid) sequence. Is it possible to clone in pET28 (a) vector in E.coli and to purify this small peptide?
I would be happy if anyone can tell me about this. Thank you
Yes, it should be possible - the easiest way to do this will be to design some overlapping oligos and anneal them, then clone them into the vector of choice.
As to the purification, you would have to tag a peptide that short to purify it (you probably couldn't even bind it with an antibody).
Although it may in theory be possible to produce it in bacteria, I would prefer to order it synthetically. Less hassle and guaranteed pure peptide. Unless you need a really big amount, I assume the cost will even be lower than if you would put all your time and effort in doing it yourself.
I have no affiliation with any peptide producing companies, it would just be my preferred course of action.
Be mindful of which codons you use for this. In mini-genes such as this you run the risk of lethality if the codons used have low levels of associated tRNA peptidyl transferases. See the following paper for an example of this. They used 2-codon minigenes but slightly longer (up to 5 iirc) can kill your cells, too. This is the idea behind NEB's cloning vector pMiniT.