Lab Report #35: From DNA to protein (part 2)

Join us in the lab to see how scientists make the proteins that players design in Foldit. In Part 2, we begin with bacterial cells and end with pure protein. If you missed Part 1, you can catch it here.

- Purifying protein designs in the lab

- A CD47 binder design by LociOiling and MrZanav in Puzzle 2172. This design packs tightly against the target binding site, and has great binder metrics. See for yourself in the Design of the Month sandbox puzzle.

(Mon, 08/01/2022 - 13:24  |  4 comments)
Joined: 09/24/2012
Groups: Go Science
Thank you for thes videos !

I've a question. After eliminated less soluble proteins and separating the right (molecular weight?) proteins by centrifugation, you say you tag our protein with histidine chains, then linking with nickel etc.
But aren't there other proteins also tagged and selected by this process?
Moreover, how can you tag and untag without breaking the protein ?

Joined: 05/19/2009
Groups: Contenders

Bruno, the tagging will have been done in the DNA coding phase, even before it was included into the bacterium. It is like adding a few residues that consist of a certain number of histidines to the end (or beginning) of your protein.
See also:

spvincent's picture
User offline. Last seen 21 min 14 sec ago. Offline
Joined: 12/07/2007
Groups: Contenders
His tags

I was wondering about those extra histidines too. Do they get trimmed off at some point? Or is there good reason to think they won't interfere with the folding of the designed protein?

rmoretti's picture
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Depends on the experiment

To some extent it depends on the experiment. Molecular biology supply companies sell a variety of plasmids where you can insert your gene to be expressed. You can get versions with or without the his tag, with the his tag at the N or C terminal, and with or without various protease recognition sequences between your gene and the his tag. Depending on which one you choose (or if you choose to roll your own), the tag can be trimmed off later or not.

One of the benefits of a His tag (versus some of the other affinity tags which one might be able to use) is that it's small - less than a dozen residues at most. Often times this is small enough that it doesn't affect the function of the protein. Additionally, it's at the N or C terminal, which for evolutionary reasons tend to be in regions where small differences in length don't matter much. Therefore, since it's easier (and because you can lose some protein during the cleavage and cleanup process) a lot of the time people don't bother to trim off the his tag.

You can see this in some of the PDB entries. The sequence records of PDB files should contain the full sequence of the protein which was crystalized. Very often you'll see a string of H's at the start/end of the protein, as the crystallographers left the his tag on when they crystallized the protein. However, those residues rarely show up in the actual structure, as the his tag is just sort of flopping around at the outskirts of the protein and doesn't have clear density.

That said, sometimes the his tag does affect the protein. Just this week I overheard some discussion about whether it could be the presence of a his tag which explained some inconsistent data about whether a protein was acting as a monomer or a dimer.

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Developed by: UW Center for Game Science, UW Institute for Protein Design, Northeastern University, Vanderbilt University Meiler Lab, UC Davis
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