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Streptavidin

Posted by Chris on February 20th, 2014  ⟩  0 comments

I believe that our current quality of life has been most affected by the discovery of vaccines, even more so than by our use of antibiotics. I also firmly believe that the benefits of those vaccines outweigh any and all side-effects, disadvantages and the scare-mongering tirades about inerudite, nonsense-driven “research”. I am personally, extremely ecstatic that I never had to worry about getting polio or mumps or rubella. Though the shots hurt like crazy at the time, I can now silently thank my parents for listening to the “advice” of knowledgeable doctors and scientists who promised them I could have a better chance of life than the one their parents lived with. t is advice that I have also chosen to listen to for my own children, rather than let them fall prey to the ravages of the most terrifying diseases and plagues that our species has dealt with so poorly throughout millennia…until only just these last hundred years or so.

Unlike antibiotics, vaccines work with our body’s natural defenses by helping to build an initial bulwark against the specific antigens of a disease that can then be repopulated at a later time in the event that the disease manifests. You can think of vaccines more like an underground, resistance force laying the groundwork for a potential invasion as compared to an antibiotic’s nuclear missile strike after the invasion already took place. That cooperative action also means that we don’t have to worry about things like developing immunities to vaccines, since they are not the main actors on the stage of combat, only the catalysts for better host immune response.

There are, of course, some inherent risks involved with vaccines. Attenuated microorganisms in some vaccines may go rogue and develop into the full-fledged disease they originated from. A person’s immune response may also go hyperactive against the vaccine dose, causing traumatic problems. In therapeutically vaccinated patients, there may not enough of an immune response left in the body (for any of a variety of reasons) so that vaccination does little to help sufficiently subdue the disease. These are not trivial matters, to say the least, and doctors and scientists are constantly working to create better and more efficacious vaccines or vaccination systems. Recently, a group from the University of Navarra in Spain, under Juan Jose Lasarte, may just have found a novel method to improve how we produce vaccines.

Lasarte’s team had already previously discovered that the fusion of the Extra Domain A (EDA) of fibronectin to an antigen leads to an increase in its association with TLR4-expressing dendritic cells, causing greater immunogenicity of the antigen and greater induction of immunity. But the fusion of EDA to every single, different antigen is time-consuming, problematic and prohibitive. But what if they could find a more universal approach to creating the fusion? Lasarte’s team focused on building a hybrid EDA-linked streptavidin recombinant protein that could be simply and easily linked to any biotinylated antigen or protein due to the high affinity streptavidin naturally has for biotin. They called their new protein the “EDAvidin”.

One of the most important aspects of the streptavidin-biotin interaction is the requirement of the tetramarization of the streptavidin protein. Through a series of tests and comparisons, Lasarte’s group could see that the new EDAvidin still formed into the tetramer orientation required for optimal biotin interaction. Even better, they biotinylated an ovalbumin (OVA) protein (using something very similar to one of Gold Bio’s Biotin Labeling Kits) and successfully pulled the OVA proteins out of solution using EVAvidin in an ELISA assay. The binding affinity wasn’t quite as good as the original streptavidin-biotin, reaching only to 10-14 as compared to the original’s Kd of 10-15, but that’s still really good! The EDAvidin also maintained the proimflammatory activity that they noted in their earlier work with pure EDA, producing an immune response in the T cells of mice similar to that of antigens linked only to EDA.

All in all, EDAvidin presents the best of both worlds: a method to produce better acting vaccines that can be ubiquitously used with any protein that can be biotinylated, which is pretty much any protein. Lasarte’s method may end up becoming the building block for an entire new system of vaccine therapy, leading to better cancer tumor control and immunoresponse. I think that it’s just another brilliant use of an already brilliant system.

 
 

Arribillaga, L., Durantez, M., Lozano, T., Rudilla, F., Rehberger, F., Casares, N., Villanueva, L., Martinez, M., Gorraiz, M., Borrás-Cuesta, F., Sarobe, P., Prieto, J. & Lasarte, J. J. (2013). A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo. BioMed research international, 2013.

Category Code: 88241 88231


 

PS. For an awesome example of what vaccinating does for us as opposed to not vaccinating, you can check out Penn and Teller explaining it in their unique, condescending (and humorous) way.

 
 

Posted by Chris on February 13th, 2014  ⟩  0 comments

In the comfortable culture of the U.S., which we take for granted all too often, it can be difficult to remember the human plagues and diseases that have followed mankind throughout the millennia. It’s difficult to remember that we, ourselves, are the preferred breeding ground for a host of bugs, worms and parasites; not just idle or incidental carriers of these microscopic beasts, but their main course…dependent on our unique physiology in order to reproduce and survive.

Some of these diseases are famous, due to their pugnacious nature or severity of effect; diseases such as malaria, botulism, or River Blindness. Others are less well known, either due to their more limited environment or because their incidence in more developed countries is less common, such as the protozoan, Entamoeba histolytica. However, less common does not necessarily mean less destructive. Entamoeba is a group of anaerobic parasites that specifically target humans and primates. Typically passed from feces and water to another host in a cystic form, these protozoans mature in our digestive tracts and cause a disease called Amebiasis. Amebiasis is typically characterized by abdominal pain, amebic dysentery, bloody diarrhea, and fevers. The protozoans can also occasionally escape the colon and end up on other organs to cause liver, brain or lung abscesses. Current estimates are that nearly 50 million people, worldwide, suffer infection from E. histolytica, which result in around 100,000 deaths. However, only 10-20% of all infections become symptomatic, so the number of infections may actually be much higher. The rate of infection of E. histolytica in tropical countries in Central or South America, Africa and Asia is actually nearer to 50%! And when the numbers of E. histolytica are combined with similar, but non-symptomatic protozoans (such as E. dispar and E. moshkovskii), the total world count may be closer to 10% or 500 million infective cases.

Protozoan immunofluroescentThe most likely place to start looking for better methods of disease prevention of Amebiasis are the surface protein interactions between the protozoan and our intestinal walls which cause adherence and activation of the protozoan to its trophozoite stage. Cataloguing and defining these surface proteins is about as easy as identifying all of the various countries on Earth from a telescope on Mars. To date, only a smattering of about 20 of these surface proteins had been identified. Iris Bruchhaus and her group from the Nocht Institute for Tropical Disease wanted to blow that number away and settle once and for all what proteins need to be studied.

Bruchhaus’ group used a non-permeable biotinylation process (similar to Gold Bio’s Biotinylation kits found here) to bind with all of the surface proteins on the HM1:IMSS strain of E. histolytica. Those biotin conjugated proteins were then easily isolated using a streptavidin agarose resin system (which you can also conveniently find here), and analyzed in NanoLC-MS/MS in order to identify the proteins. They found close to 700 proteins! Oddly though, nearly 50% of the proteins found they found did not show any specific membrane association. Bruchhaus’ group was able to further detail many of these isolated oddball proteins, showing that roughly 85% of them did have some cell surface interaction, even if not in the traditional sense. But this actually implies that the plasma membranes (and their surface associations) of these protozoans are not static and easily defined at all, but are a very dynamic, complex and interconnected weaving of molecules in constant exchange on and across the membrane.

That doesn’t necessarily make this type of work any easier. But with a large number these proteins identified, work can at least begin on some of the most important proteins, further characterizing the association with their human host cells. Who knows, maybe it’s actually one of these oddball proteins that only sometimes associates with the plasma membrane that account for the low percentage of the protozoan becoming symptomatic. Time will tell…

 
 

Biller, L., Matthiesen, J., Kühne, V., Lotter, H., Handal, G., Nozaki, T., Saito-Nakano, Y., Schumann, M., Roeder, T., Roeder, E., Krause, E., & Bruchhaus, I. (2014). The Cell Surface Proteome of Entamoeba histolytica. Molecular & Cellular Proteomics, 13(1), 132-144.

Category Code: 88241 88231

Posted by Chris on January 22nd, 2014  ⟩  0 comments

As I continue to search for novel and interesting applications for the interaction between biotin and streptavidin (check out my earlier blogs on bacteriobots and coral symbiotes), I love seeing the variety of applications that scientists from around the world dream up and develop! Most recently, I came across a paper describing a new method for DNA detection and fluorophore quenching using streptavidin-coated gold nanoparticles.

Chuan-Liang Feng et al. developed a system which utilized colloidal gold nanoparticles (AuNPs) to act as a DNA biosensors, or DNA probe, in order to detect DNA hybridization. Colloidal gold has been used for over a thousand years in various industries, starting with the glass blowers of ancient Rome and Greece who produced some truly amazing and beautiful, colored glass using gold particles dispersed throughout the glass to create light diffraction effects. One of the best known examples of this level of technology is the Lycurgus Cup (click here for another interesting article concerning this magnificent work of art). In our modern era, however, colloidal gold has become popular in the medical field, being investigated extensively as a drug carrier or in this case, gene therapy.

Streptavidin Gold Particle DNA detectionFeng’s novel approach in labeling the AuNPs with streptavidin and using its natural interaction with biotin to create a specific probe. Even more interesting, Feng used the association of biotin/avidin to build small, single-strand, complementary (or near complementary) DNA probes with either a biotin or Cy5 tail. Amazingly, the association, first between the biotin-ssDNA and the StAuNP, and the between the StAuNP/biotin-ssDNA and Cy5-ssDNA created a quenching effect on the fluorescence of the Cy5 molecule! Further, Feng’s group was able to add fully complementary, non-biotin ssDNA probes to the solution and slowly recover Cy5 fluorescence, outcompeting the near-complementary biotin-ssDNA and thus reversing the quenching effect!

This method is a simple and effective means to detect DNA hybridization, and as Feng reports, could be used as a high throughput method of biodiagnostics. Further tests will tell. One thing is for certain, scientists will continue to dream, create and develop new and ever-more fascinating systems to increase our understanding of the world we live in. And I, for one, cannot wait to see what we discover next.

 
 

Feng, C. L., Dou, X. Q., Liu, Q. L., Zhang, W., Gu, J. J., Zhu, S. M., Jenkins, A., & Zhang, D. (2013). Dual-Specific Interaction to Detect DNA on Gold Nanoparticles. Sensors, 13(5), 5749-5756.

Category Code: 88241 88231