Strep-tag affinity chromatography protein purification - theory & practice

In affinity chromatography, the proteins you want selectively stick to the resin, while the proteins you don’t want flow through. And then you push off the proteins you do want with a competitor to get them all by themselves. But of course, you can’t just will the proteins to stick or not stick, you’ve got to stick to the biochemistry! The protein you want has to have something that none of the other proteins have, and which the column likes to bind specifically. Thankfully, with recombinant protein expression this is “easy” to achieve - since we’re providing the genetic instructions for making the protein (recombined into a piece of DNA called a vector which we stick into cells in a flask to make it for us), we can add a bit on to those instructions to add a bit onto the end of the protein that can serve as an “affinity tag” - and one of my go-to tags is the “strep tag”
full text & figures: bit.ly/strepta...
This particular strategy is based on biotin-avidin/streptavidin interactions. Those are some of the strongest known non-covalent and thus “easily reversible” interactions and we can harness their power to help us purify proteins by sticking a biotin mimic as a tag on our protein and binding it to little beads coated with a streptavidin mimic.
When you’re purifying a protein with an affinity tag you need the tag to bind to the resin strong enough that it’ll stay bound when you wash the column to remove non-specific binders. But the interaction has to be weak enough that you can get your protein unstuck without harming it. Biotin binds streptavidin so strongly that the conditions needed to get it off are so harsh they’ll probably hurt your protein
So, instead of sticking “real biotin” onto our proteins and binding it to “real” streptavidin, we use modified forms.
For the biotin mimicking part, I use a “Strep Tag.” The Strep-tag is an 8 amino acid (protein letter) sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) (WSHPQFEK). In terms of amino acid qualities, this sequence is pretty well-balanced so it doesn’t affect the properties of the protein like its overall charge.
I use a Twin-Strep-tag (WSHPQFEKGGGSGGGSGGSSAWSHPQFEK) which has that bindable sequence twice with a linker in between them. Since glycine’s really small and flexible (it’s side chain is just an H) the tag can contort itself so that both sites can bind a single bead (kinda like the His tag we looked at last week did). But unlike with the His tag, where each bead only had 2 spots open, the Strep-Tactin resin has 4, so it can bind more. More on the this His tag: bit.ly/histidin...
Instead of sticking it on after the protein’s been made like what happens when proteins get biotinylated inside of cells, it gets put on as part of the protein because we add the instructions for it in front of or at the end of the instructions for our protein that we stick into cells to make it for us.
And speaking of sticking things places, we need the other half of our “velcro” - the streptavidin mimic. The resin I use is a modified form of streptavidin called Strep-Tactin that binds less tightly (so more reversibly). Like always, this is NOT an endorsement, it’s just what our lab uses. The Kd of the Strep-Tag II to Strep-Tactin is about 1μM (10⁻⁶) compared to that Kd of ~10⁻¹⁴ of biotin to streptavidin.
I usually bind in “batch mode” - add lysate to resin & let it spin on a hotdog spinner in the cold room for ~30 min before column-izing it by pouring it into an empty plastic/glass cylinder with a filter at the bottom and letting the liquid flow through (trapping the resin and the bound protein)
Then I wash it with the same buffer the protein’s in and the resin was equilibrated in. This washes off anything that’s just kinda randomly stuck on.
So at this point I have my protein mostly by itself, but stuck to the beads and I need to do the shoving. Biotin could easily compete off our protein but then we still have the problem of needing to get the biotin off the column so we can use the resin again. So we turn to a slightly less tight binder that’s still a great competitor - desthiobiotin. As the “desthio” part of the name indicates, desthiobiotin is biotin minus the sulfur.
Then I add the elution buffer (which has the competitor, desthiobiotin). I let ~ 1/2 a CV (column volume) flow through (e.g. if I had 10mL resin, I’d let out ~5mL which is the liquid that was in the column before the new liquid got there). I collect this as my elution 0 (E0) - it shouldn’t have much of my protein but it’s good to keep & check.
After letting that through, I close the stopcock and give the desthiobiotin a chance to find & bind the strep-tactin (pushing my protein off). Then, after ~15 min I open the stopcock to let the liquid out (E1). Then I do more elutions with the elution buffer, collecting fractions I can test for the presence of protein

[@krishanuchoudhury7943]

I just like the way you teach and also the way you discuss!

[@thebumblingbiochemist]

Thanks so much!

[@richardmariadasse2209]

informative