If you ask a cell to make a protein for you and it tries to refuse – don’t give up yet, try to to fuse! But what fusion partner should you choose to use? I’m a little late for Valentines Day, but let’s talk about finding partners – FUSION PARTNERS! Because some proteins don’t want to go it alone. They need a friend to help them express themselves, protect them from harm, & stick with them as they try to find their “best self”. And they can get sum o that friendship from SUMO.
When we express proteins RECOMBINANTLY, we stick a gene for a protein into an easy-to-work-w/circular piece of DNA called a PLASMID VECTOR (this is the recombining part) then stick that into cells (often harmless bacteria or insect cells) to make the protein for us (this is the expression part). We’re in control of the DNA we put in there, which means we’re in control of the sequence of amino acid protein letters in the protein that gets made. But the expression cells are in control of whether that protein actually gets made – and made correctly.
To make this correct construction more likely, and to make it easier to separate our protein from all the other stuff the cell makes, we can take advantage of the thing we *are* in control of – the DNA sequence. We can add extra DNA letters to add extra amino acids (protein letters) to the end(s) of the protein. A couple things we commonly add are affinity tags and fusion proteins.
Affinity tags give your protein something un-naturally unique so that you can get it to bind something that none of the other stuff will. In affinity chromatography you take a mix of proteins and other cellular content and flow it through a resin (little beads) coated with something the affinity tag has specific affinity for – (e.g. streptavadin for strep-tag or glutathione for GST). Tagged proteins stick, untagged don’t, then you add a competitor to push your protein off.
properties of a good affinity tag
✔️ binds something that can be attached to little beads (resin)
✔️ that binding’s reversible
✔️that binding’s specific (only tagged proteins bind it)
✔️ that binding’s high-affinity (your protein sticks & stays stuck until you’re ready to remove it after washing the other stuff off)
To make sure the tag doesn’t interfere with our protein, we can tether it with a flexible linker & & we can design the linker to have a sequence that a site-specific endoprotease (protein scissors) will recognize and cut at so we can cut the tag off after it’s served its purpose. (I’m currently waiting impatiently for the tag to get cleaved off the protein I eluted from the strep column…)
That’s all great, BUT you can’t purify protein if you don’t have any, and sometimes your protein needs some help being made properly, Fusion partners are small proteins you tack onto the end of your protein to help your protein get made in the first place and stay happy. It’s kinda like a “foot in the door” marketing technique – bribe cells to start making something they like making, and then trick them into making your protein that you’ve attached to that thing they like. They fold really quickly & help your protein fold properly & stay soluble & safe.
properties of a good fusion partner:
✔️ expresses easily
✔️ folds quickly
Some fusion partners include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, and ubiquitin (UB). But the one I use the most is SUMO. GST is used a lot and it’s cool in that it can also serve as an affinity tag (more here: http://bit.ly/2LoRzOg ) but I don’t like to use it because it also has affinity for itself – it pairs up to form dimers. So instead I use SUMO.
SUMO stands for Small Ubiquitin-like MOdifier. SUMO is 100 amino acids (protein building blocks) long (~8 kDa) but for some reason it runs as if it were ~20 kDa in an SDS-PAGE gel (a way to separate proteins by size & visualize them. more here: http://bit.ly/37N2GZj It has a compact, globular core with flexible ends – it comes with its own linker!
Speaking of ends… Proteins are chains of amino acids. The sequence of amino acids in the chain is determined by the gene. The gene (DNA form) is copied into an RNA version (mRNA) (transcription) then that mRNA is used as instructions to link together the right amino acids. The amino acids are linked (w/help of ribosomes) 1 at a time to the end of a growing chain, going from N terminus to C-terminus.
The chain starts folding as it comes out of the ribosome, so the N-terminus gets a “head start.” We usually express fusion proteins at the N-terminus meaning that it’s translated before your protein its attached to so it has a chance to fold first. And it folds really quickly – SUMO’s one of the fastest folding proteins out there -the molten globule hypothesis proposes that this lets it act as nucleation site for folding of your protein
Fusion proteins promote *correct* folding of the protein they’re attached to which is really important for SOLUBILITY! SOLUBILITY is whether each molecule of something is fully coated in water. Properly folded proteins should be soluble (or embedded in a lipid membrane if they’re a membrane protein). Proteins are able to be soluble because they fold so that parts of the protein that like to interact with water (HYDROPHILIC) are on the outside & parts that don’t like water (HYDROPHOBIC) are sequestered away in the center. more on solubility (something we crystallographers think about a lot): http://bit.ly/2SJrdGR
If a protein doesn’t fold properly, those water-hating hydrophobic parts get exposed to water & panic – so they stick to the hydrophobic parts of other misfolded proteins (because that’s better than being next to water) -> they clump up & aggregate. This is a helpful way to think about it, although technically, it’s more like the water molecules don’t like them so the water molecules do whatever they can to link to each other instead or your protein, which forces the hydrophobic protein parts together – this the hydrophobic exclusion effect and you can learn more about it here: http://bit.ly/351PZby
Fusion tags help the proteins fold properly so this doesn’t happen, so your protein is soluble. A few ways how they might help:
- because they’re soluble, & they’re made first, they help keep the protein soluble as it folds (the SUMO wants to stay surrounded with water & it drags your protein with it – harder for your protein to aggregate so it decides to try hiding its hydrophobic parts by refolding instead)
- chaperoning – bind to aggregation-prone folding intermediates (earlier-translated parts still waiting for their partners to emerge from the ribosome) and prevent their self-association – it also might recruit other chaperones present in the cells to help out
- repelling other molecules &/or “hiding your protein” giving your protein more personal space to fold properly without distractions
Fusion tags can also protect against degradation such as by taking your protein to “safer” parts of the cell like the nucleus, where there are fewer proteases (protein-cutting enzymes) to worry about. & they can also enhanced expression, potentially because the fusion part’s so efficiently translated – gives your protein a sort of translational “boost”
Another great thing about SUMO is that it will leave if you ask it to – there are SUMO-specific proteases that you can add to cleave it off. And instead of just recognizing an amino acid sequence (which could, if you’re unlucky also occur in your protein) it also recognizes the shape of SUMO so it will only cut where it’s supposed to. And it doesn’t leave anything behind.
SUMO’s actually a natural thing – Your cells use SUMO attachment (SUMOylation) as a way to “flag” proteins for things like transport to different parts of the cell – In eukaryotic cells (most things except bacteria), SUMO can be added naturally as a POST-TRANSLATIONAL modification – this means that the instructions for it are NOT in the gene for the protein. It’s added AFTER the protein is made (translated). (a more familiar form of post-translational modification is phosphorylation, which we discussed here: http://bit.ly/2CkhBvD )
When it’s added post-translationally, it can be at various locations throughout the protein (it likes certain Lyses) and it’s not on *all* the protein. We want to make sure ALL of our protein gets the SUMO tag where we want it, so we attach the sequence for SUMO to the sequence for our protein in the plasmid -now SUMO is added DURING translation so whenever our protein’s made, it’s on there. And since it’s made first, it’s more like every time SUMO’s made, our protein’s on there – which is great since the cells like making sum o that SUMO!
if you’re having trouble with expression or solubility and fusion partners don’t do the trick, you might need to consider using a different expression system: http://bit.ly/2G5N5tY
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