From gene to gel – hope it goes well! Pure picked protein production from start to finish (today focusing on the start & finish). Yesterday I focused on the purification part, but before you can purify the protein you want you have to get cells to make it for you. And after you purify it you want to make sure you really did – and store it safely. 

Today’s mostly pic-based, but if you want more detail, there’s a link at the bottom to a list of past posts by topic. And I’m also working on adding blog versions to my blog (link in profile), but it takes a while so patience appreciated. You can find the long versions of things on expression & chromatography there now.

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 to posts with more about them.


  • first you need to get a hold of a copy of the protein’s genetic instructions in the form of cDNA. The c stands for Complementary. And it distinguishes this from gDNA, the Genomic version. gDNA is the original, unedited gene as it lives in the genome (collection of all your DNA). This gDNA has a lot of extra regulatory information that isn’t used for protein-making, so when the cell makes a messenger RNA (mRNA) copy of the gene to give to the protein-making machinery, it edits this information out.
  • cDNA is a DNA version of this mRNA, so it’s basically the edited gene. Often you can buy cDNAs for different proteins from plasmid repositories & you can then put it where you want by “cutting and pasting” with restriction enzymes & ligase or “copy and staple”ing with PCR based methods like SLIC.
  • speaking of plasmids, the next step is to put the cDNA into a plasmid you want. Moving a piece of DNA from one plasmid to another is referred to as SUBCLONING. Often we put the cDNA into a plasmid where we can induce overexpression (tell the cells when to start making protein & have them make a lot of it)
  • you want expression to be inducible for a couple reasons – firstly, because before you have them make protein, you want to check that the cloning actually worked, so you want cells that will make lots of copies of the plasmid and not waste their time making protein you don’t even know if is good yet.
  • you also want expression to be inducible because when you tell the cells to start making tons of your protein they devote most of their resources towards that and stop dividing. So you need to let them grow and divide to a nice population size first. You can monitor this by measuring how cloudy it is.
  • so the first thing you’ll want to do is put your newly-made plasmid into a different strain of bacteria, a “cloning strain” that’s “high copy number” meaning it will make lots of copies of your plasmid. Getting the plasmid in the bacteria is called TRANSFORMATION & we usually use “heat shock” where we sensitize cells with calcium chloride, drench them in plasmid & then heat them briefly to open temporary holes so the plasmid can sneak in
  • then we let them recover and grow and make lots of plasmid we then purify out (we call this a mini prep) and send it for sequencing to see if it really worked. We use antibiotic selection (plasmid has antibiotic resistance gene that plasmid-less bacteria has) to weed out cells without the plasmid, so we know the plasmid’s there but not whether the right gene is


  • for bacterial expression, we usually use a T7-based over expression system where we induce with IPTG which lets the cells make mRNA & protein

PURIFICATION – PROTEIN CHROMATOGRAPHY is a method used to purify proteins by taking advantage of their different properties by sending them traveling through a CHROMATOGRAPHY COLUMN – which is just a tube filled with little beads (RESIN).  

I like to think of it kinda like sending people through a museum that takes them different amounts of time to go through and catching them as they come out. They go in the entrance (top of the column) and out the exit (elute from the bottom of the column) where you can use a UV monitor to see where protein comes out (like tracking when people exit). 

Proteins absorb at 280nm so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. And the computer shows this to us as a peak. more here:

But *all proteins* do this, not just the one you’re interested in. So it can tell you how many people are coming out at different times, but not whether they’re Bobs or Joes or a mixture. As they come out we use a fractionator to divert them into different wells in a deep-well plate based on when they come out (like putting everyone who comes out of the museum between 4 & 4:15 in one room, 4:15 to 4:30 in another, etc.

to see what’s really there we use SDS-PAGE (Sodium Dodecyl Sulfate- PolyAcrylamide Gel Electrophoresis). here we basically want to erase all the “unique” features of proteins we’ve been exploiting so we can directly compare their lengths (number of amino acids, or molecular weight (m.w.)). but we only do this to a tiny amount of our protein – just enough to see

throughout the purification process, we’ve been careful to protect our protein – even if that means working in the cold room (the bumbling biochemist’s nemesis). with SDS-PAGE, you still don’t want your protein to get chewed up, but you do want it to get unfolded.  in the ion exchange step, we relied on the protein’s natural charge. but with SDS-PAGE we hide their natural charges and give them a coat of uniform NEGATIVE charge that will allow us to attract them to a POSITIVELY charged electrode at the bottom of the gel. In SEC, we relied on the protein’s natural SHAPE – which is related to length but not directly – think of a flat versus crumpled versus really crumpled piece of paper. 

If that looks ok, you want to freeze your protein (but not the water inside & around it. If you want to PREVENT something from FREEZING, don’t get it cold… or ADD SOLUTE! 🤗 We call solutes we add to prevent freezing CRYOPROTECTANTS 👍

Water’s weird in that it EXPANDS when it freezes 🤯 MOST LIQUIDS DON’T! Water does bc it freezes in an orderly lattice (which solute “gets it the way” of 👍) in which, in order to achieve optimal binding angles, it has to spread out

⏩ if water freezes inside pipes, they can burst 😬 & if it freezes inside our protein, they can “break” 😬😬 So we add CRYOPROTECTANTS to protect pipes in winter & proteins when we freeze them 

What to add? 🤔👇

FREEZING POINT DEPRESSION is a COLLIGATIVE PROPERTY (as are vapor pressure depression & boiling point elevation), meaning it’s the # of dissolved particles, not the identity of the particles that matters 👍

So you might think, why not add an ELECTROLYTE like NaCl (table salt) which dissociates into its component ions (charged particles) when you dissolve it (e.g. (Na⁺ & Cl⁻), giving you more particles than you put in? 🤷‍♀️ 

You could, & this is why we salt our streets to prevent them from icing over💡But salt comes with a high charge – literally!⚡️ Because it breaks into charged particles, it can have undesirable consequences (like breaking up IMFs in our protein not just the water!😬) or corroding metals 😬

We want to add something that’s more “water-like” in terms of not disrupting the system (but less water-like in terms of “stickiness”) 👉 Basically, we want something kinda “blah” – and SMALL so we can pack lots of it in there 👉 small polyols (chemicals with multiple -OH groups) like GLYCEROL fits the bill & are commonly used as CRYOPROTECTANTS 👍

Note: When we froze the cells, we didn’t add glycerol – instead the freeze-thawing was an important helper in breaking the cells open. 

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