Henry Jakubowski



I am also initiating a new project derived from my sabbatical research in Dr. David Bernlohr's lab, Department of Biochemistry, St. Paul, MN., and in continuing collaboration with him. Briefly, by using modern techniques in molecular biology, including site-specific mutagensis, I have prepared to variants of a protein found in human fat cells (adipocytes). The protein, adipocyte lipid binding protein (ALBP), binds fatty acids in an water-filled internal cavity. The activity of this protein in the cell is regulated by phosphorylation of tyrosine 19. In the phosphorylated state, the protein will not bind fatty acids. However, if a fatty acid is bound to the protein, the protein becomes phosphorylated in the cell at a greater rate than if the fatty acid is not bound. These apparent contrary findings suggest that conformational changes occur in the protein upon phosphorylation. I have prepared by mutating the gene for the ALBP protein a mutant in which the tyrosine is replaced by aspartic acid. The aspartic acid should mimic the electrostatic environment of the normal (wild type) protein in the phosphorylated state. One goal of the project is to characterize the properties of the mutated protein produced after transforming E. Coli with plasmid DNA containing the mutated gene, and expressing the mutant gene. Bacterial were transformed with the gene, and expressed the mutant protein. However, the protein proved to be insoluble and stored in inclusion bodies in the bacteria. I have purified these inclusion bodies, and have significant amounts of the insoluble proteins. The next step in this project is to develop methods to solubilized the inclusion body proteins, and refold them to a native state. Once this is accomplished, additional studies to characterize the protein will be made.