Internal A2E synthesis capability created and protein purification improved
Milestones achieved in 2010: The creation of an internal A2E synthesis capability enabled efficient target enzyme evaluation. Our screening and development of several protein purification protocols resulted in the isolation of an active, A2E-degrading enzyme that is engineered to have features that make it interesting from a clinical perspective.
One of our priorities at the SENS Foundation Research Center has been to overcome the limitations of our ability to test potential A2E-degrading enzymes caused by a restricted supply of the target molecule (A2E). We have now successfully synthesized A2E, purifying it by flash chromatography and HPLC, and confirming it to be identical to that produced in Janet Sparrow's laboratory through different analytical tests. Our ability to produce this material "in house" is a key element in the expansion of our screening and evaluation of both existing and newly-purified A2E-degrading enzymes.
In collaboration with students at the State University of New York at Plattsburgh, we have also made significant progress in dramatically improving the A2E-degrading capacity of one of our enzymes after fusing it with a second enzyme. While the former catabolizes the target molecule, destroying its toxic activity, the latter produces a metabolite that accelerates the degradation activity up to a hundredfold.
The year ahead: We will express and purify additional candidate enzymes for both macular degeneration and atherosclerosis projects, exploiting new biochemical strategie; establish activity for these enzymes in vitro; perform lysosomal-uptake studies in RPE cells for macular degeneration and in macrophages for 7-ketocholesterol; and perform initial toxicity studies.