Arking:JCAOligoTutoria20
Translational Fusions
One of the primary motivations in developing the BglBricks standard was to facilitate the construction of fusion proteins. The underlying principle here is that proteins tend to fold in modular units called "domains". Some proteins are composed of a single domain, while others contain multiple domains. These folding units are often also functional units, and it often it is possible to recombine two such functional units from different sources into a single molecule and preserve the activity of both functional units. To explain this further, let's take a look at an example. Shown below is the crystal structure of an IgG protein and below that is a simplified representation of the IgG protein. IgG proteins are one of several forms of antibodies that are made within your body as key components of the immune system.
Image from www.physics.purdue.edu/people/faculty/nolte.shtml
In the diagram above, the IgG protein is colored based on its polypeptide chains. There are 4 polypeptides in the single IgG protein: two heavy chains (in blue) and two light chains (in red). Each oval in the diagram represents a single folding domain. All the domains that make up antibody proteins have a type of fold called the immunoglobulin fold, and these domains are referred to as immunoglobulin domains. All the information to make a properly-folded polypeptide is encoded within each immunoglobulin domain. So, if you constructed a coding sequence (CDS) for a single immunoglobulin domain and provided an acceptable promoter, ribosome binding site, and terminator in the right places, you could express a properly-folded immunoglobulin domain within a cell. Now, that would be a properly-folded domain--not necessarily a fully-functional antibody. In fact, one immunoglobulin domain itself would be pretty useless. To understand this, let's first examine what IgG proteins do. Primarily, they bind to a particular "epitope" which is usually a short peptide often from a virus. Additional proteins within the immune system also interact with the IgG protein and induce responses that result in the destruction of the bound species.
So, the IgG molecule is able to do lots of things in the context of your bloodstream. The most basic of these activities is its ability to bind to its epitope. The epitope is bound in the cleft between the VH and VL (V="variable") domains of the IgG. In practice, the minimal component of the IgG protein that retains the binding ability is called the Fab fragment which contains 2 polypeptides, each of 2 domains (two variable domains and two "constant" domains, CH and CL). One might expect that just the VH and VL domains together might be sufficient to get the binding activity. In practice, the affinity of VH and VL for one another is fairly low, and expressing these two domains as separate proteins is usually not sufficient to obtain a functional binding protein. However, it is possible to construct a fusion protein, referred to as a single chain antibody, or "scFv" and get a single polypeptide that retains all the binding activity of the original IgG molecule.
Even more interesting, one can fuse other polypeptides that have nothing whatsoever to do with the immune system to the scFv molecule and obtain a functional polypeptide that is the sum of the scFv protein's activity and the activity of the protein fused to it. So, for example, an enzyme can be fused to the scFv and the product would both bind to the epitope and also perform the chemical reaction of the enzyme. One of the most common things fused to scFv proteins is the pIII protein from M13 phage. The pIII protein lies at the tip of the M13 phage particle. By fusing the scFv to pIII, it is possible to generate M13 phage particles that display the scFv on their surface. This technology has been used extensively to engineer antibodies that bind to altered substrates and is called "phage display".
Now, constructing a fusion protein is not truly an act of "gluing two proteins together". In reality, what you are doing is constructing a DNA encoding the fusion protein. To explain this, check out the graphic at right that illustrates how phage display looks
"Fusion proteins" are proteins that are composed of more than one source proteins combined into a single polypeptide. They are highly related to "chimeric proteins"
By this, what we mean is that you may want to use standard assembly to encode proteins that are themselves composite parts of individual subunits. One of the most common applications of
