Showkat Ahmad
How can we express the viral protein that is not complete ORF (i.e. with out start or stop codon) but a part of one complete gene and becomes functional protein after Post translation modification ( i.e poly protein that results in more than one proteins that work individually)?
Hi Showkat,
Simply clone the fragment that you want to express into a bacterial expression vector. You will have to add a start codon in front of the fragment and a stop codon at the end, there's no way around this as far as I know. The resulting protein/domain/peptide will have an extra amino acid at the N-terminus (the start codon will code for a M), which is unavoidable but hopefully won't affect the overall structure of the peptide you're trying to express.
Many expression vectors can add a tag for affinity purification or for solubility to your protein. Most of the proteases used for removing these tags generally leave one or two extra residues on your protein. If you definitely can't live with the extra M from the start codon or the extra residues left after removing the tag, then you can add: a) MNNNNK, or b) MIEGR or c) MIDGR in front of your sequence, which are the preferred cleavage sites of Enterokinase [for a)] or Factor Xa [for b) and c)], which leave no residues. After you express the protein this way it will have 5 extra residues at the N-terminus, but you can remove these with the aforementioned proteases, which will cleave your protein after the K or R residues. I haven't used either of these proteases, but I know that Factor Xa is not very specific and it might potentially cleave your protein at several other sites that contain basic residues, leaving you with several fragments instead of the single long peptide you want.
Greetings... Antonio Ariza
Thank you very much for your response..i think this is on of the best idea..
Thank you once again :-)
Just to add to what Antonio has already said, most bacterial expression vectors will have protease cleavage sites built into them. This allows you to express your domain/protein of choice with a purification tag then remove said tag with the protease.
The pET and pGEX series of expression vectors are very popular and have various tags N- and C terminal with different protease sites to choose from. Start codons are found in both nde1 and nco1 restriction sites making it relatively easy to PCR the gene of choice with a nde1 or nco1 site in the primer should you wish an C terminal tag.
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