From DNA to Protein, Step 3

Okay, this is it!  The last step!!  As always, review the previous posts in this series if you’d like to understand more of what is going on here.  

Let’s remember what we did in Step Two: Bacteria are the best tool scientists have for making protein.  One little bacterium is not sufficient for making as much protein as we need.  In fact, scientists typically grow liters worth of bacteria.  While the bacteria grows and is churning out its own proteins necessary for survival, it is also making our protein of interest: GST-RED.  Eventually, we collect all the bacteria, which are full of GST-RED, and freeze it until we’re ready to finish this whole thing out!

BACKGROUND/EASY EXPLANATION

                This part is like cooking in that the scientist needs to understand subtlety.  The broad strokes of what I’m going to explain to you will read like a recipe, but any chef knows that grace, control, and keen understanding is necessary to actually make delightful meal.

                With that in mind, let’s get that pellet of E. coli bacteria out of the freezer.  YUM.  In case you are wondering – no, it doesn’t smell good.  After working in a lab of 12 – 18 people (depending on the time of year) and being surrounded by growing bacteria every day, the smell really doesn’t bother me anymore.  However, if you aren’t used to it, it can seem downright disgusting.  

                  The pellet is individual bacterial cells filled with GST-RED.  The first step to getting to the GST-RED is to break open the bacterial cells.  Several ways exist to do this (sonication, cell disruption, French press, lysozyme) so I’m not going to bore you with the details.  Instead, I’m just going to tell you it can be done!  A visual diagram of this step can be found in Figure 46.1.

                Remember, breaking open cells is like bombing the walls of a house.  Everything was nice and organized inside and then becomes a downright disaster.  Everything you don’t want is also mixed up with what you do want.  Following the breaking open of the bacteria, we are left with a great goop of stuff that includes DNA, fats, and all the other bacteria proteins in addition to GST-RED.  How in the world do we separate everything from GST-RED?  HOOOW?

                The first step is through centrifugation.  By spinning this mess at high speeds, anything very heavy is going to straight to the bottom of the tube to form a pellet.  GST-RED is not something that will be found in the pellet, but things like broken membranes and large DNA pieces will be.  Awesome – we’ve gotten rid of some of our problems!   To visually see this step, check out Figure 46.2.

                The second step is to use something called resin.  Resins can be bought at a variety of companies.  They are composed of small, gel-like beads that are linked with a particular compound.  In our situation, our resin will have beads that are connected to a compound called glutathione.  It’s not important what glutathione is beyond the fact that the GST within our GST-RED protein loves to bind to glutathione. 

If we take our centrifuged sample and mix it together with this resin, GST-RED is going to bind to it but everything else should not.  The other proteins do not have GST and have no interest in glutathione.  To see visually see this step, look at Figure 46.3.

Once all the GST-RED is bound to the resin, it is washed a few times to get rid of any other remaining proteins.  Then you are left with the left hand side of Figure 46.4.

The last step is to just add a small amount of elutant to the resin.  Elutant is something that either the resin likes more than your protein or your protein likes more than the resin.  In either case, the protein is kicked off the resin and the scientist collects it in a tube.  If all goes well, you have successfully achieved the final box of Figure 42.1: only red triangles.

Congrats!  We’ve made it!

Because of the nature of this step, there are many many many factors to consider.  I’m going to skip the more complicated explanations of anything because I could be writing all day.  If you have any specific questions, please feel free to ask!

From start to finish, this entire protocol of Steps 1 (preparing DNA), 2 (growing cells) and 3 (purifying protein) takes about two weeks.  Step 1 is usually the longest: it lasts about a week to go from nothing to confirmed plasmid.  Step 2 takes ~ 24 hours.  Step 3 takes anywhere from 24 – 72 hours.  It really depends on what kind of fanciness needs to be done to achieve purified protein.

Do you think you can do it now??

REFERENCES

Me, myself and I