Hello RG!
I've designed a construct that will be expressed off a plasmid. I would like to add some restriction sites so I can swap out parts of the construct later on. My concern is adding restriction sites (6 nt = 2 amino acids) will impact protein folding. Does anyone have experience with this? Should I be concerned? Or will the two amino acids encoded by the restriction site not have enough pull to affect folding of my 400 amino acid peptide?
Thanks!
Dear Stephanie
it really depends from where you add this 2aa and which are the 2aa.
if you add this aa in a flexible linker beetween your protein and possible fusion tags probably the folding of your protein will be not affected.
try to add the 2aa to your protein sequence, predict 2ndary structure and check if the presence of the news aa affect in some way the structural elements ( alpha elics or beta sheets that are close to it in the protein sequences).
if no changes are observed, its a good sign.
However i would like to reccomend you to evaluate PIPE cloning which is an enzime free cloning methods that leave you Free to the use of restriction site and using it, you do not have to add nothing.
you can find information about PIPE cloning on my blog:
https://proteocool.blogspot.com/
at PAGE1 Cloning and expression
good luck
Manuele
There is some non-zero risk of that. It's not a risk one should take in the 21st century. Restriction cloning is obsolete. You should make all your variations by de-novo synthesis. It's even much cheaper than cloning, considering the value of your time and the risk of cloning not working right away. Also, as Manuele wrote, it's worth learning a restriction-independent cloning method in the longer run. Gibson Assembly would be another one worth looking at.
I agree with the others, you can't ensure that it won't affect protein folding and, therefore, have unintended consequences on protein structure and function. I can recommend the pCoofy cloning system, which is a parallel SLIC cloning method, meaning that you can use the same primers with the same restriction sites to generate a whole host of constructs with a wide variety of tags, cleavage sites, and expression systems (from E. coli over Insect cells to Mammalian cells). At that point, if you want to truncate your construct on either end, you just order a primer with the same overhang and the same restriction site but the consensus sequence that corresponds to the C- or N-terminus of your construct, amplify, ligate, and transform. No need for restriction digest.
One thing about pCoofy you have to keep in mind is that you can't happen to have the restriction sites they use anywhere else in your construct, so it is possible that you will need to perform site-directed mutagenesis to perform silent mutations that remove the restriction site but keep the peptide sequence intact.
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