Mercury Detoxification Protein Structures

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Purification Time and Blog Rules. Fused.

Well, this is my last post, even though I feel like telling the world about various steps of purification, growth, and NMR techniques in detail. But, since it is a big world with New Media best at work, it is really not necessary for me to put up purification protocols on my blog. I'm not the master of purification, but I know enough to allow me to work on my own when the time comes (actually, I have been working solo through my graduate student's project for the last two weeks). So, to end this blog, I will try and dissipate my thoughts into providing you guys with a sense of timeline for the entire purification process.

Purification of the protein takes a total time of two weeks per batch. And each batch yields about 8 milligrams of protein (it's not much, but that's all is required for NMR analysis). Different proteins have different yields, but I will stick to my protein, MerF. So, we start off with our growth and protein expression, which takes an entire day. Second step is lysing the cells and performing column chromatography (chromatography is a technique of separation of chemicals and in this case, separation of MerF and everything else in the cell), which takes another entire day. The rest of the steps, which I unfortunately didn't have the time to explain in many different blog posts, take up the entirety of the next two weeks. I will briefly explain them below in the right order:

Dialysis - My sample is added into a dialysis bag, which has pores of a certain size. The main goal is to not let my protein out of the bag. So the pore size will be smaller than the protein size. In dialysis, I exchange out everything except the protein, again. This takes about 2-3 days.

Lyophilization - I freeze-dry the sample in order to dissolve in a different solution. This takes one day.

Cyanogen Bromide Cleavage Reaction - The protein is attached to another protein (usually called Fusion protein), which helps in the column chromatography. This fusion protein is not necessary any more and is cleaved via a reaction. This takes another day and is followed by dialysis and lyophilization. These take up 3-4 days combined.

The final step is High Pressure Column Chromatography or HPLC. HPLC is used to separate the protein from the fusion protein that remains in the sample. Before HPLC, the purity of the sample is 50% our protein and 50% fusion protein. After HPLC, it is ~98% our protein. HPLC is a big machine that does things on it own after loading our sample. This takes one day.

So, if we add up all the days, we have 11 days in total, but not everything goes according to plan, so we have hiccups every now and then, which causes a delay of a day or two, bringing the time to about 13 days in total - one day shy of a fortnight. There, I have briefly explained the purification timeline in as simple terms as possible. 

Now for the blog rules. I have established some sense of fear and doubt into the non-scientific reader's mind, all unintentionally of course. But, below are a few guidelines that I have tried to follow in most of my blogs.

To try and stay on topic for most of the blog post as in, not change the topic entirely midway to explain a different process that could have been done in an additional blog post.

To type up descriptions of various process of purification steps in simple terms and with analogies, if possible, to make it easier to understand for the non-science reader.

To address the same audience with every blog post. Technicality trends upwards, but slowly as you start reading a number of posts. This is normal for any scientific blog. Once you are engrossed, technicality goes up eventually since you learn to pick up on terms through Google searching or Wikipedia :P

To pick up on news in my field through different blogs or scholarly articles that I may find through other blogs.

To include a link or a picture in every blog post if it may help understanding the nature of the concept.

These are really meant as a guideline as opposed to a set of strict rules. Sometimes, they are effective in presenting a given topic, sometimes not. But, adhering close to these guidelines would help the reader and blogger to present coherently. And with this, I end my blog and hopefully, I will have matured my knowledge enough within the next year to start a real purification blog or website. 

Thank you for reading and hope you didn't have a headache trying to understand the technical terms. :D

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Bacterial Cells and their Growth

So, in my last post, I had discussed about the system of protein expression. Now, I will talk about what kind of bacterial cells we use and what kind of specifications we use to grow them. So, the bacteria we use in our protein expression system is Escherichia Coli or as it is usually seen as E. coli. These are rod-shaped bacteria that are found in the intestines of warm-blooded animals. Most E.coli strains are harmless, but some can be toxic and are known to cause serious food poisoning in humans. Below is a picture of E.coli under a microscope. 

E.coli bacteria have the capability of surviving in conditions where other bacteria cannot, so this bacteria is the prevalent choice for researchers when conducting biological research. Also, the genetic data aka DNA is very simple to modify, making it easy for researchers to modify it to their heart's content.

In the lab, these bacteria are grown in media that is rich in nutrients and vitamins (literally) at about 37ºC, so they can proliferate without any problems. And antibiotics are added to prevent any other kind of bacteria to grow since the media is very rich for any other micro organism to grow. One of the many media, called LB Media, contains table salt, yeast extract, and Tryptone. Yeast extract and Tryptone are a nitrogen source (or providers of amino acids), which is essentially what the bacteria need to grow. From the first post, amino acids are the foundation for muscle growth and body functionality (for humans and any other organism). Bacteria grows till a good amount has grown and then they are lysed (cell lysis is the process of breaking up the cells without compromising the integrity of the contents of the cell. It takes about 1 hour for bacterial cells to double their volume and we let them grow for about 4-6 hours before they are lysed. And then the process of purification of the desired protein starts, which is a topic for another blog post.

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Process of Protein Production

So, let's talk about how protein production works. As I have said in my previous post, protein production starts when mercury ions bind to the MerR. Also, when the cells have to express proteins, they tend to undergo a lot of stress. When doing research, we cannot risk exposure to mercury ions every time we need a sample for analysis. In other words, we will not risk our lives, to that extent, in the name of science when there are other alternatives around. So, instead of mercury ions present, we use a generic molecule which induces production of all proteins in a cell continuously till stopped by the researchers. This generic molecule is Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG.

IPTG binds very tightly to MerR (or any other protein regulator). Usually, any kind of generic inducer is degraded by the cell to prevent over-expression of proteins and relieve the cells from a lot of stress. So, the IPTG binds tightly to the receptor in a way that prevents the cell from degrading it and stopping the production of proteins. 

The above image is a cartoon representation of what is happening in the cell for a different protein, but the only thing different is that GFP is one block, and in our cells we could have that block for the production of MerT, MerF, MerP, MerR, MerD, MerA,  or MerB. The green arrows mean that the expresion of protein starts from there. LacI, which is present in almost all cells, is the natural repressor to prevent the cell from going into over-expression of proteins and stress. This repressor comes off when there is abundance of inducing molecules that bind to it, for e.g., excess mercury ions or IPTG. The IPTG then binds to LacI and the expression of protein starts from then till the inducer molecules are not present or the researcher stops the cells from growing.

In the next blog post, I will talk about what kind of cells are used to produce these proteins and possibly how their DNA is modified in the lab.

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Mercury Detoxification Family of Proteins

            This family of proteins helps in detoxifying reactive mercury. A mercury atom is reactive if it has one or two electrons less than its atomic (stable) state. Reactive mercury is usually called ions. When mercuric ion is present in the air or water, it reacts with other molecules to satisfy its electron deficiency, i.e., it reacts much easier to gain electrons. Anything that has too many or too few electrons is much more reactive than the correct amount that it needs. These mercuric ions are highly toxic.  and are released into the air by industrial factories. Measures can be taken to limit the amount of mercury ion being released, not all of it can be contained. At the moment, our bodies have no self-defense mechanism to detoxify mercury ions. We have to depend on external chelating agents that bind mercury and transport them out of our body.

            This family of proteins has been found in many different single-cell bacteria. There are four different types of proteins involved in the process of converting ionic mercury (too few electrons) to atomic mercury (the right amount of electrons). The first one is the transporter (MerP in picture), which selectively binds and transports ionic mercury from the outside of the cell to the specific ion channels (MerC and MerT in picture), which are the second type of protein. Ion channels are like one way traffic lanes that only let some type of vehicles pass, i.e., in this case, mercury ions. The ion channels let ionic mercury come inside of the cell and lead it to the Reductase (converter) (MerA and MerB in picture), the third type of protein and by it’s name, it converts ionic mercury to atomic mercury. It is the biggest protein in the family and is the most interesting for our study. The fourth type of protein is the regulator protein. This protein regulates the production of transporter and reductase proteins depending on the amount of mercuric ion captured by the transporters. If more and more ions are present, there is up-regulation by MerR, i.e., more production of reductase and transporter. If the amount of mercuric ion decreases, there is down-regulation by MerD. The picture below is a summary of the family of proteins. HgX2 and CH3HgX indicate ionic mercury as toxic molecules and Hg0 as atomic mercury. 

  Picture taken from Barkay, T., Miller, S. M. and Summers, A. O. (2003), Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews, 27: 355–384. doi: 10.1016/S0168-6445(03)00046-9

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Biological Research as Practicum

My practicum research was on the determination of protein structure using Nuclear Magnetic Resonance (NMR). I will explain to you what NMR is in a later blog post. Let me tell you about proteins. Proteins are large, complex molecules that are present everywhere in our body – in cells, in tissues and organs, and even the bloodstream. They play a critical role in transporting minerals to cells and tissues, in catalyzing reactions by enabling them or driving them faster, in messenger activity by coordinating different tissues in the form of hormones, and even bind to foreign bacteria and viruses to prevent them from infecting our bodies.

            Now that we know what functions proteins play in our bodies, let me tell you what they are made of. Proteins are made of smaller molecules called Amino Acids. They are not what we generally call acids, it’s just a name given to them. There are 20 different types of amino acids that are connected to each other in long chains. These long chains can be hundreds or thousands of amino acids. The sequence of these amino acids is what determines the unique, 3-dimensional structure of proteins. My research was to determine the same 3-dimensional structure of the protein. Below is an image of the basic structure of amino acids.

            There are two types of proteins – Integral and Peripheral. Peripheral proteins are usually attached to domains of the Integral proteins. Integral proteins are usually attached to the walls of cells. There is a later of phospholipids(not necessary to know right now), which are long chain molecules. The picture below is a better explanation and summation of everything I just said. The proteins lie from outside of the cell, through the phospholipid membrane, and to the inside of the cell. The protein that I am working on is a transmembrane protein, which helps in transporting Mercury from the outside of the cell to the inside for detoxification. There will be more on that in the next blog.