Arking:JCAOligoTutoria22

Generating parts encoding circularly permuted proteins

A circularly permuted is generated by (conceptually) linking together the N and C terminus of a protein into a circular molecule, and then cutting it back open at a different site. In the DNA, what this boils down to is doing something like this:

Note that in this example, I've shown how you would permute a periplasmically-expressed protein, hence the prepro sequence targeting it to the periplasm. When permuting, regulatory features need to stay in the same spots--you should want to "spin around" the active peptide. If this protein weren't periplasmic, it would be even easier to permute. You'd just flip the N and C terminal regions and that's it.

To illustrate how this done, let's try making one! Let's design a circularly permuted T4 Lysozyme. I have no idea whether the product of this construction file is a functional protein or not, but you'll get the idea of it. First of all, grab the crystal structure of it. T4 Lysozyme (T4L) is heavily studied structurally, so there are tons of files available on pubmed or pdb. Let's look at PDB ID: 3DN1.

The first question to ask is where are the N and C terminus. Are they reasonably close to each other? If not, you're probably not going to be able to make this work. They don't have to be right on top of each other, you can make up for some distance with a flexible linker. They look pretty good in T4L. You also want to look for a place to cut it back open. The ideal spots are going to be large disordered loops. T4L doesn't really have one, so we'll just go with one of the loops. Gly51 looks like a reasonable spot. There are more sophisticated modeling tools that probably would be wise to use for this sort of design, but I won't get into that here.

To make this thing, we need a template DNA. Berkeley iGEM 2008 has cloned T4L already and also removed the restriction sites, so we'll start with pBca1256-K112012, which you can download here.