Henry Jakubowski
THE COLLEGE OF SAINT BENEDICT/SAINT JOHN'S
UNIVERSITY
ALBP RESEARCH PROJECT
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.