I thought it had been only a day or two since my last entry. But four days? Whew.

I'm still in a rather bad habit of going in to work in the middle of the night, so the days sort of smoosh together after a while. Combine that with Beth cramming for her finals, and you get the idea what it's like in the Ihms household these days.

Oh yeah, and let's not forget my job situation.

I'm not here to gripe about my problems however, I just wanted to share something pretty cool that happened last night.
For the last several months, I've been trying to separately purify the three protein components of the circadian clock, the proteins KaiA, KaiB, and KaiC.
This particular time I was in the last stages of purifying KaiB.

Most people think that purifying a protein is all black magic and hocus pocus. Nothing can be farther from the truth. I'm going to go into some detail, not to confuse you, but to show how really simple all this stuff really is.

First of all, you start with a circular bit of DNA, called a plasmid. Now, among other things, this plasmid, or "vector" contains four items of note.

1. A lac operator. This is a sequence of DNA to which a lactose inducer (a protein) binds. The protein is part of the normal lactose pathway of the bacteria we will eventually put the plasmid into.
2. A T7 promoter. The T7 promoter is a bit of DNA which basically "recruits" the protein expression machinery (the ribosome and whatnot). The name T7 comes from the T7 bacteriophage (a virus) which upon infection of bacteria, hijacks the cells protein machinery to make it's own proteins.
3. A gene that codes for a protein that makes the bacteria resistant to a antibiotic of our choice.
4. Our gene that, upon translation, will make our Protein.

Here's how it works:

1. The plasmid is put into the bacteria in a process known as "transformation".
2. Bacteria are grown till mid-log phase. This means that the bacteria are dividing as quickly as they ever will.
3. IPTG, a non-hydrolyzable lactose analog, is added.
4. The aforementioned lactose pathway protein (mentioned in number 1 in the "parts" list above) binds to the sequence when IPTG is present.
5. This "induces" the bacteria to start making our protein.
6. The T7 promoter, being very good at recruiting the translation (protein making) machinery of the cell, causes our protein to be made at a very rapid rate.

If you've been paying attention, you will want to know what the antibiotic resistant gene is for. Well, when we transform the bacteria with the plasmid, not all the bacteria pick it up. As a matter of fact, only a small percentage do. Therefore, to select for only the bacteria that do have the plasmid, we "plate out" or grow the bacteria in media containing the antibiotic. This causes any bacteria not having the plasmid (and therefore the antibiotic resistance gene) to die, and only those blessed with the gene to live.
Now of course, even this isn't as simple as that, because it's fairly common for bacteria to delete our gene, and just tote around the resistance gene, which is obviously a pain for us. This can only be detected when we go to induce our bacteria, and nothing happens.

Anyway, once we have the little bacteria buggers making our protein, we let them do that for several hours. Since IPTG cannot be hydrolyzed, it stays in the media and continues to "induce" the cells to continue making our protein of interest.
It's interesting to note that in KaiC's case, this mechanism actually works too well. The cells make so much protein that KaiC is no longer soluble in the cell's cytoplasm, and so it precipitates inside the cells, forming inclusion bodies. Because protein precipitates are generally unfolded, this is worthless to us. To get around this, we grow bacteria that are expressing KaiC at a low temperature, and induce weakly with only a little amount of IPTG.

Once the protein is no longer being made (basically because all of the bacteria are dead from exhaustion), we spin down (centrifuge) the cells. The cells are then resuspended in what's called a "cracking buffer" or lysis buffer.

The cells are the lysed by passing them through a french press. A french press is basically a big piston. Inside the piston chamber, you have a very high pressure, about 1300PSI. There is a small valve at the bottom of the chamber, and as the cells go though the valve, the difference in pressure causes them to pop. (Hence the lysis).
Once the cells have been "french pressed" two or more times, they are re-centrifuged to remove all the celluar debris. At this point the protein should be soluble in the supernatant.

Now here's where the fun begins.

When we make a plasmid containing our gene (that codes for our protein), we usually design it with several "add-ons" that will help us purify it later. One of this is a hexahistidine tag.
A hexahistidine tag is nothing more than six histidine amino acids in sequence. It has been discovered that when this many histidines are in a row, they act as a fairly effective chelator of metal ions (they can bind to them).

Why is this useful? Well, think about it.
A bacteria is very complex. There are literally thousands of different proteins in the cell. Granted, if the bacteria expresses our protein well, we should have 10X more of our protein than any other proteins in the cell, but still, we need to separate this one protein from all the others.
How do we do this? With the hexahistine (or 6His) tag.

We take the "crude lysate", or the liquid left over after cracking the cells, and we pass it over a MAC, or Metal Affinity Column. This is a column that has metal ions (of our choosing) stuck to it. Therefore, when we pass the crude lysate over the column, only our protein will stick to the metal ions, which are stuck to the column matrix material. (In theory).
To get our protein off of the column, all we have to do is pass imidazole (basically the active part of histidine) over the column. This frees up our protein, causing it to come off all at once.
Neat, huh?

What's really scary is that while I was at IWU, I envisioned something similar. Since I hadn't really read any biochem journal articles, I didn't know that a 6His tag (or anything like it) existed. I envisioned a simple ligand, or group that one could attach to a protein to vastly increase it's affinity to a column, allowing for selective purification. It was a great idea, and could have made me wealthy beyond imagination, but I was only 15 years late. Oh well. The funny thing is, I actually wrote about this theory on several Grad School applications, where I'm sure they thought I was a numbnut.

Continuing. Well, we now have our fairly pure protein. In theory, a MAC is all you would ever have to use, and you'd have a single protein by the gallons. Unfortunately, it doesn't work as simply as that. Often, we need to run several columns after the MAC, usually a anion-exchange, a size exclusion column, or both.
To make matters worse, many times we have to cut off the six histidines to make our protein active. This involves adding another protein that has a cut site specific for the region around the hexahistidine tag. After cutting, you then have to purify the protein further.

See? No black magic, just cold hard theory and lots of work.
Now imagine this: I can take a batch of bacteria, and one day after induction, I can have pure protein ready for cutting. And I'm not the fastest one in the lab, either.

Back to my original story.
I've completed all the steps except the very last column, when I forget to actually even hook the stupid thing up. As a result, all of my protein, my precious, time-consuming protein, goes straight down the drain. No possibility of recovery. CRAP!

Well, as you can imagine, I was quite upset. However, I remembered that I still had two fractions left over from my MAC column (I had screwed up several steps past that column).
So, I went back, and using these fractions, attempted to re-purify. I tell you, the Good Lord was looking out for me, and after much supplication, I got my protein. What a trip.

And that, ladies and gentlemen, is a day in the life of a protein chemist.