Protein purification day! Despite having to face my nemesis – the cold room – it still makes me feel yay! I’ve done a lot of protein purifications during the past 3 years of my PhD journey. Like a lot a lot. But it had been 3 whole weeks since I did my last one (I’ve been too busy testing out the pure protein from the past preps)! I think that’s the longest stretch I’ve gone without purifying for probably like a year or so… I do a ton of protein purifications because I’m trying to figure out how these proteins work. And, thanks to RECOMBINANT PROTEIN EXPRESSION I can “tweak” the genetic recipes for them, stick those recipes into cells to make them for me, and then purify them out using PROTEIN CHROMATOGRAPHY and test them to see if the tweaks tweaked the tested thing. 

Since I can make lots of tweaks, I can make lots of different “constructs” of the same protein. Today I did the first steps of purifying 3 such proteins (it took me a long time to work up the courage & confidence to do multiple at once – especially since the proteins are just really similar versions of one another so you can’t easily see if you swapped them! careful labeling is key!) 

You know how you often get asked what a typical day for you’s like? Well, there is no “typical day” for the bumbling biochemist because I’m always doing different things – like one day I’ll be purifying, and then I’ll be doing different types of experiments. It’s one of the things I love about my work. And there’s no real “typical” protein purification because it varies based on the protein’s preferences, but here’s a general idea of how a protein purification might go…. How do we select what stays and what goes? Is the prep going well? How can I tell? Disappearing bands on an SDS-PAGE gel

I’m a protein biochemist so, as a “molecular mechanic” of sorts I study how proteins work (or don’t work). Which requires me to have lots of pure protein, so I do a LOT of protein purification. I’ve done a lot of posts on various aspects of the process, but here I want to give you an overview of the entire process.

Each protein’s different, so the process has to get tweaked & optimized for different proteins. Here I’m just going to take you through the process of producing and purifying a simple, cytoplasmic (e.g. a water-soluble protein that lives in the general “main part” of cells & isn’t embedded in a membrane) protein through recombinant expression with a His tag in bacteria (if these words aren’t familiar, don’t worry, I’ll explain all these terms).

We can break up the workflow into 3 main parts:

  • -MOLECULAR CLONING – this is where we take the gene (DNA instructions for making the protein) from its original home and put it into a VECTOR PLASMID (a small, manipulatable, circular piece of DNA) and put that vector into (harmless) bacteria
  • EXPRESSION – this is where we have the bacteria make the protein for us (we can also express trickier proteins in insect cells or mammalian cells
  • PURIFICATION – this is where we break open the cells & isolate the protein we want from all the other stuff. It usually involves several types of PROTEIN CHROMATOGRAPHY, where we use columns filled with little beads (resin) that separate proteins based on their different properties (charge, size, etc.)

Each of these main parts has multiple sub-parts. I don’t have time/space to go into them all in detail here, so I’m going to provide links (at the bottom) to posts with more about molecular cloning & expression and, in this post I’m going to focus on the purification phase. Each substep of this phase serves to remove different things from your protein of interest 

HARVESTING – this is where you remove the liquid media (bacteria food) from the cells after they’ve made your protein, but leave the cells intact. You do this by pouring the cell-filled media from the flasks you’ve grown them in into bottles and centrifuging them (spinning them really fast) – the cells are heavy so the sink to the bottom, forming a pellet. Then you can pour off the liquid (supernatant), resuspend the cells in a cleaner liquid & flash freeze them in liquid nitrogen & store them in the -80C freezer until you’re ready for the next step

LYSIS – this is where we actually break open the cells – we thaw the pellet, add lots of salt to disrupt the membranes and sonicate them to use waves of energy to break up the DNA so it doesn’t gunk stuff up

ULTRACENTRIFUGATION – this is where we separate the membrane pieces from the soluble stuff. It uses much higher speeds than the centrifugation we did before because the membrane pieces are much lighter than the whole cells (though still heavier than the dissolved proteins so the membrane bits pellet while your protein remains in the liquid part (supernatant). So here you want to keep that supernatant.

At this point, we call the supernatant the LYSATE. And it has your protein, but also a lot of other proteins. You can see this if you run an SDS-PAGE gel, which separates proteins by their size. more here: http://bit.ly/2GZc3tG

To get rid of those other proteins we use PROTEIN CHROMATOGRAPHY. More here: http://bit.ly/30LklxG

I start with an AFFINITY CHROMATOGRAPHY step. This uses resin that recognizes something really specific – usually an affinity tag we’ve added onto the end of the protein (by putting the genetic instructions for it before or after the instructions for our gene in the plasm). Because affinity chromatography is recognizing something “unnatural” and highly specific, it can (hopefully) remove most contaminating proteins – but not all.

A common affinity tag is a His tag, which is just 6 or 8 Histidines (a specific amino acid), which will bind to a Nickel (Ni) or Cobalt (Co) coated column (Immobilized Metal Affinity Chromatography; IMAC). Other proteins have Histidines too. But not that many in a row, so your protein will bind preferentially. It’ll hog the column and the other proteins will flow through.

Then you need something that will outcompete the His tag to get your protein off. Bring on the imidazole. It looks like His, so you can flood the column with imidazole to push the His-tagged proteins off. But before you flood it, you wash it with low levels of imidazole to remove non-specific binders that are just binding cuz they happen to have a lot of Hises.

Once you’ve eluted your protein (gotten it to come off the column) you have the option to cut off that tag using an endoprotease (protein scissors) that recognizes a sequence between the tag & the start of the protein.

Once you’ve removed the tag there’s nothing “artificially super specific” about your protein, so you now have to exploit natural differences between your protein and any remaining contaminating proteins (which now includes that protease you added).

The first other property we’ll exploit is charge. Proteins have different charges because they’re made up of different combinations of amino acid letters, some of which are charged. more here: http://bit.ly/2C2od2y

We’ll exploit this using ION EXCHANGE CHROMATOGRAPHY (IEX), where we bind proteins to resins that are oppositely-charged. Ions are charged things and basically you “exchange” ions from salts (like the Na+ or Cl- of NaCl (table salt) with protein ions. Then you can gradually increase the salt concentrations so that those salt ions outcompete the protein and you get another exchange. Or you can change the pH to change the protein’s overall charge (the lower the pH, the more free H+ for the protein to latch onto -> become more positive & vice versa)

In CATION EXCHANGE chromatography you have negatively charged resin & you’re binding & exchanging positively-charged (cationic) proteins & salt ions.

ANION EXCHANGE chromatography is the opposite – you have positively charged resin & you’re binding & exchanging negaatively-charged (anionic) proteins & salt ions.

At this point your protein is hopefully pretty darn pure. But proteins can have similar charges, so there are likely still small levels of lingerers. They might have similar charges but chances are (hopefully) they’ll have different sizes. So next we can use SIZE EXCLUSION CHROMATOGRAPHY (SEC)(aka GEL FILTRATION) to separate the remaining proteins by their size. In this type of chromatography the resin’s “boring” to the proteins so the proteins don’t interact with it. But the resin’s also “bored” in the sense that it has “secret tunnels” “bored” into it. The tunnels have different diameters so proteins have to be small enough to fit in order to go through them. So the smaller the protein, the more tunnels it will go through and the longer it will take to go through the column. In this way, the proteins get separated by size, with bigger things coming out sooner, smaller things later.

Often, SEC is considered a “polishing step” because your protein’s usually mostly pure going in. But even if it doesn’t have enough contaminating proteins to cause problems, it also likely is swimming in a lot of salt. Another reason SEC is useful is that it acts as a buffer exchanger. When your protein comes out it’s in the buffer you’ve been running through the column.

Now you (hopefully) have really pure protein! You should see one nice band on your SDS-PAGE gel.

For long-term storage of the protein, you want to keep it at -80C, usually with a cryoprotectant like glycerol to prevent harmful ice crystals from forming. more here: http://bit.ly/2U8XwRo

Proteins don’t like ice forming and they also don’t like being frozen and woken up and frozen and woken up and… You want to avoid multiple freeze-thaws, so you freeze “single-use” aliquots. This is probably my least favorite part of the purification process because if you have a lot of protein it can be really tedious.

My hand was getting super sore from all that tiny writing on the top of the tube (I like to label the tubes with the construct number, concentration, & date), so I got some printable cryodot labels. That helped, but now I keep finding random cryodots on my clothes in the laundry!

After aliquoting, we flash freeze the aliquots in liquid nitrogen (flash freezing gets the water in and around them to freeze in place without time to link up into ice crystals) more here: http://bit.ly/2OpBBGi

His-tags work well for bacterially-expressed proteins, but the proteins I’m purifying today were expressed in insect cells. Insect cells and cells from other eukaryotes (things with membrane-bound rooms inside their cells to house their DNA, etc. – so basically most stuff except bacteria) have a lot of proteins that naturally have a lot of Hises. So instead of the His tag I’m using a “Strep-tag.” more here: http://bit.ly/2lFw1SE

But basically it mimics biotin & binds to a column that mimics streptavadin – the biotin/streptavadin interaction is one of the strongest non covalent (non-electron-sharing) interactions known so these mimics are weakened a little so the protein doesn’t get stuck to the column forever & we can elute it off with another biotin mimic, desthiobiotin. Bacteria naturally have a lot of biotinylated stuff so strep tags don’t work as well for them, but this isn’t as much of a problem with insect cells

You have to give the endoprotease some time to cleave the tag off, so I’ve set up the AKTA (a protein purification system that’s like a subway system for buffers – you can direct different liquids to flow different places so you can automate the chromatography) to run while I sleep :). If all goes according to plan I should have cleaved, eluted protein waiting for me in the morning so I can continue on to SEC! Fingers crossed!

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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