We have talked about making cDNA libraries and antibodies before but what happens if you generate an antibody to a protein have no way to clone it using a nucleic acid probe. In this instance, you can use your antibody to clone the encoding cDNA by making what is called an expression library. Expression libraries are very similar to more traditional libraries but instead of screening for the DNA of interest, the bacteria transcribe and translate YFP and you use an antibody probe to find the colony of cells that have expressed YFP. The trick then is to get the bacteria to express all of the cDNA in the library and for this we have the bacteria make a fusion protein.
Figure 1. Diagram of a generic expression vector, reading frame #1.
An expression vector is like any other plasmid with a polylinker for cloning into, an origin of replication, and an anitbiotic resistance gene (figure 1). The polylinker is located in the lacZ gene and there are three slightly different plasmids, one for each reading frame. The goal is to clone the cDNA library into all three reading frame versions of the expression vectors so that your favorite cDNA will be in frame with one of the three expression vectors. When your cDNA is in frame, it will encode the amino acids for YFP which you want. The resulting protein is called a fusion protein because you have fused ß-galactosidase with YFP (figure 2). Although neither half of the fusion protein will probably be functional, all you need is an epitope for your antibody to bind. So you make three cDNA libraries, induce the bacteria to express the plasmid encoded proteins and go looking for your epitope.
Figure 2. Fusion protein which contains some b-galactosidase and all of your favorite protein.
Now that you have made your expression libraries, it is time to plate them out (figure 3). You get lots of petri dishes with the appropriate medium and antibiotic, and spread out the bacteria so that when they grow to form colonies, they do not touch each other (for the most part).
Figure 3. Two views of a library that has been plated and colonies are big enough to see.
Each colony is made of millions of genetically identical (clonal) bacteria that are derived from asexual reproduction of a single cell. The bacteria contain four main components that you need to think about (figure 4). 1) The cell wall and cell membranes, 2) proteins (both normal bacterial proteins and the fusion protein), 3) bacterial chromosome, and 4) plasmids containing the cDNA which encodes YFP.
Figure 4. Bacteria contain proteins (pink), plasmids (light blue), and chromosomal DNA (dark blue).
Now that the colonies are grown and the cells have expressed all fusion proteins (not only YFP fusion product but all others encoded by all the different cDNAs in the library), you need to get those proteins onto a membrane and off the agar medium. The classic membrane is nitrocellulose, though there are many other options available. The membrane is placed on top of the bacteria and all the proteins and nucleic acids stick to the membrane (figure 5).
Figure 5. A circle of nitrocellulose is lower onto the bacteria and used to lift them off the petri dish.
The membrane is flipped over so that the bacteria side is up and placed into a solution which will 1) lyse the cells and 2) denature all the proteins (figure 6). Now the bacteiral proteins are stuck on the membrane and ready to be probed with your antibody.
Figure 6. Top panel, the bacteria are lysed open to release their cotents which adhere to the membrane (bottom panel).
You should notice that the pattern of lysed cells is a mirror image of the colonies as they grew on the plate (figure 7). This is cause by putting the membrane down and then flipping it over. Nevertheless, you now have a replica pattern of all the bacterial colonies that contain one of many possible cDNAs.
Figure 7. The pattern of lysed cells (left panel) is a mirror image of the bacterial colonies (right panel).
The membrane is placed in a protein-containing solution that blocks the membrane so there are no "sticky" places left on the membrane. The blot is placed in a solution that contains your antibody which can be detected (figure 8) either by radiation (usually 125 I, but this method is not very popular any more) or by enzymatic detection usually by the production of light in a process called chemiluminescence (the production of light by chemical means - breaking of covalent bonds).
Figure 8. Cartoon showing an antibody binding to a particular protein (pink) and not binding to other proteins or nucleic acids (light and dark blue). The antibody is covalently modified to carry an enzyme that will generate light when incubated with the appropriate substrate.
The antibody finds and binds to its epitope and since there are many copies of your favorite fusion protein, and many copies of your antibody, enough light is given off to expose X-ray film (figure 9). Once the X-ray film is developed, you can line up the film with the plate and pick some of the remaining bacteria off the plate. These bacteria contain many copies of one expression vector that has cloned into it a cDNA that encodes an epitope that your favorite antibody binds to.
Figure 9. The top portion shows the alignment of the X-ray film with the membrane which has been incubated with your antibody and treated with a substrate that will allow the enzyme to generate light. One spot appears on the film which indicates which spot of lysed bateria contains your protein (as a fusion product). The X-ray film used to determine which colony contains the cDNA encoding YFP.
So in the end, you have used an antibody to clone a cDNA that encode the protein you want to study. At the most basic level, there are only two ways to clone a piece of DNA. Either know something about the sequence and use a nucleic acid probe, or utilize the form/function of the proteins to work your way back towards the encoding DNA the way we did in this example. We will see that there are many other clever ways to clone DNAs of interest, but they are merely variations on one of these two themes.
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