I'm trying to clone and express a protein in E.coli using pET26b or 33b vector. I am designing primers using the NcoI and XhoI restriction enzymes. I want to know how to design primers to include the C-terminal His tag in my protein, i.e. how to make the reverse primer or choose the restriction enzyme so that the his tag is in frame with the protein. Also, when I attach a his tag at C-terminal, will the extra 2-3 amino acids from the vector sequence also be expressed attached to my protein C-terminal?
Hi Avinash,
What you will need to do is to design the 3' primer to include six additional histidine residues, and then a stop codon. It is usual to use alternating codons for the histidines, and the TAA stop codon is the most commonly used. So, your final construct will look like this:
last codon of your protein - His - His - His - His - His - His - Stop - Xhol
with the sequence:
Your sequence - CAC - CAT - CAC - CAT - CAC - CAT - TAA - CTCGAG
You need to consider whether the C-terminus of your protein will need a short linker to make the His-tag accessible (usually a short GSGSG linker would be appropriate, although most times it is unnecessary), and whether you need the His-tag to be cleavable (most times it doesn't help, as most protease sites are as big as or longer than the His-tag). If either of these situations applies, then that will affect your primers, obviously: most likely they won't.
Then, you need to design the primer that will act as a reverse of this. Clearly, this will depend on how you do your cloning (i.e. do you cut the PCR product directly - in which case adding a few random bases beyond the restriction site is a good idea; or put the product into an intermediate vector and then subclone). You will also need to have a longer sequence identical to the sequence of your protein, as your artefact for cloning is going to be nearly 30 bp - so you probably need 30 bp at least identical to your sequence.
This worked for me with pET26b.
I hope that this helps!
Hi Avinash,
What you will need to do is to design the 3' primer to include six additional histidine residues, and then a stop codon. It is usual to use alternating codons for the histidines, and the TAA stop codon is the most commonly used. So, your final construct will look like this:
last codon of your protein - His - His - His - His - His - His - Stop - Xhol
with the sequence:
Your sequence - CAC - CAT - CAC - CAT - CAC - CAT - TAA - CTCGAG
You need to consider whether the C-terminus of your protein will need a short linker to make the His-tag accessible (usually a short GSGSG linker would be appropriate, although most times it is unnecessary), and whether you need the His-tag to be cleavable (most times it doesn't help, as most protease sites are as big as or longer than the His-tag). If either of these situations applies, then that will affect your primers, obviously: most likely they won't.
Then, you need to design the primer that will act as a reverse of this. Clearly, this will depend on how you do your cloning (i.e. do you cut the PCR product directly - in which case adding a few random bases beyond the restriction site is a good idea; or put the product into an intermediate vector and then subclone). You will also need to have a longer sequence identical to the sequence of your protein, as your artefact for cloning is going to be nearly 30 bp - so you probably need 30 bp at least identical to your sequence.
This worked for me with pET26b.
I hope that this helps!
Hi Avinash,
I agree with Nicholas on the design of the sequence. In terms of the primer design, you can make one large reverse primer with the his tag etc as the overhang but, in my experience, the gene specific portion of the primer should be a bit more than the (about 1.4 times works well for me) the size of the overhang which makes a very big primer of about 70 bps! This could work but if not then you can also try to create the His tag etc using 2 sets of reverse primers - the first primer containing the first 4 his residues then the second primer containing the remaining 2 his residues, stop codon and RE site. But remember the gene specific portion of the second primer needs to contain the his residues of the the PCR product using the first rev primer. This way you do 2 PCRs:
(1) the first PCR using fow primer and rev primer 1
(2) use the PCR product from (1) as the template for the 2nd PCR using the fow primer and rev primer 2.
I hope this makes sense. Good luck
Dear all
Thanks a lot for all the help. The pet26b vector already has a optional C-terminal Hist tag, so I think I could design a reverse primer with XhoI and place it in frame with the his tag. I just want to know if the his tag and the amino acids that get added to the C-terminal (from the vector sequence) in the protein will have any adverse effects on protein folding and function or cause problems in structure determination. Especially since I may not be able to cleave the his tag..
Hi Avinash,
it depends on the size of your protein and its active sites. Is it close to the C terminal? However, this His tag is very small and, in most cases, should not interfere with the protein folding or function. I recently expressed a 15kDa protein where I included a TEV cleavage site and a 10x His tag downstream of the protein. The protein had the same level of functionality whether cleaved on not
Dear Dhadchi,
Can you be a little more detailed ? like how much was your primer length and Tm. Also, I believe you should have read Anil's suggestion as well. Instead of one, two primers should work better in terms of Tm limits.
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