Erin Wu and David Erickson, Microbiology & Molecular Biology
Introduction
Antimicrobial chemokines is a type of host defense protein that play an important role in the natural defenses of many organisms such as defending skin and mucosal tissues against bacterial infections.These peptides have a net positive charge, which contributes to membrane permeabilization and/or cell penetration. It has been observed that CCL28, an antimicrobial chemokine, binds to the surface of bacteria, however, it is not known how they bind, or what they do, to cause bacterial death. Research has demonstrated that Yersinia pseudo tuberculosis with mutations in lipopolysaccharide Oantigen and core synthesis genes (rfaD mutation) exhibit increased binding affinity for the antimicrobial chemokine, CCL28. This suggests that the surface proteins and the lipids exposed might be binding targets for this chemokine. My lab has shown that when the defective core oligosaccharide (rfaD mutation) bacteria was treated with proteinase K, an enzyme that degrades surface proteins, there was diminished binding affinity for CCL28 as compared to the untreated cells. My goal in this project is to identify the surface proteins that are potential targets for CCL28.
Methodology
The first procedure was extracting the membrane protein of Yersinia pseudotuberculosis.This was done by using the french pressure cell press to lyse the cells and then isolating the membranes by centrifugation. The membrane proteins are then incubated with CCL28, which is indirectly bound to beads. In many of the procedures that we conducted, we saw a high number of nonspecific binding of membrane proteins to the beads instead of CCL28, which gave us unreliable results. To prevent this from happening, the membrane proteins were incubated with unbound beads and washed several times. After the nonspecific binding proteins are separated from membrane proteins that bind specifically, CCL28 was added to the washed membrane proteins and incubated for 2 hours at 4˚C. Since the result of centrifugation causes denser substances settle on the bottom of the tube and substances that are less dense to the top, we were able to separate the CCL28 bound membrane proteins from the unbound membrane proteins. To verify our results, we ran them on an SDSPAGE gel.
Results
The SDSPAGE gel on the right illustrates the results of this experiment.
Lanes (Left to right):
- Membrane Protein (untreated)
- CCL28 Only
- Leftover Beads
- Nonspecific binding of membrane protein and beads
- Unbound membrane proteins (0μL CCl28)
- Bound membrane proteins (0μL CCl28)
- Unbound membrane proteins (1μL CCl28)
- Bound membrane proteins (1μL CCl28)
- Unbound membrane proteins (5μL CCl28)
- Bound membrane proteins (5μL CCl28)
- Unbound membrane proteins (10μL CCl28)
- Bound membrane proteins (10μL CCl28)
Discussion
Several procedures were designed to try to isolate nonspecific binding of membrane protein from the ones that bind specifically. As can be seen in lane 4, much of the nonspecific binding proteins have been isolated. The expected results are bands that are present in the bound membrane protein lane would not be present in the unbound membrane protein lane and visa versa. However, there are no differences between the lanes of unbound and bound membrane proteins, even with the varying amounts of CCL28 added. These results suggest that there are still binding between non specific proteins and the beads in the specific membrane binding mixture. Therefore, we are unable to identify the potential protein targets of CCL28.
Conclusion
Our main problem of this experiment was unable to isolate the nonspecific binding membrane proteins from the membrane proteins that bind specifically. Further purification of these membrane proteins will be able to prevent nonspecific binding of proteins and beads from happening. When this happens, we will be a step closer to identifying the specific proteins that are targeted by CCL28. This will bring us closer to understanding the basic mechanisms of the mechanisms of the innate immune system, which could lead to potential discovery of novel treatments.