(1068-A) Development of Biochemical and Cellular Assays to Monitor Bilirubin Levels

Abstract: Metabolites are important descriptors of cellular state and so can be used to monitor the phenotype of cells and animals.
Heme is an essential cell metabolite, best known for being required for hemoglobin, but also being required for various cellular processes including electron transport. Bilirubin is the end product of the degradation of heme; resulting in a more water soluble metabolite that can be secreted from cells and animals. Bilirubin is thought to have a major role as an antioxidant helping to protect cells from oxidative stress.
Previous reports have described the protein UnaG, from Anguilla japonica, which becomes fluorescent upon the binding of bilirubin so that this can be used to monitor bilirubin levels in a homogeneous, continuous manner. However, our initial efforts to use this protein were hampered by low levels of protein expression and solubility. To increase protein expression a Maltose Binding Protein fusion was created and evaluated. The fusion protein not only showed greatly increased expression in E.coli cells but the fusion protein showed greatly enhanced aqueous solubility, which greatly facilitated handling and ease of use of the protein.
The greatly enhanced solubility, and apparent protein stability, then made it possible to develop a number of assays to monitor bilirubin levels in biochemical and cellular formats.