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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Christopher
Herrington (Vanderbilt University) (Marcia Newcomer, LSU Dept.
of Biological Sciences) Overexpression of Retinal
Binding Enzymes
The biologically active forms of retinol or Vitamin A are extremely
important in gene regulation and development in embryos. The
two active derivatives of retinol are its oxidized forms, which
are retinal and retinoic acid. Aldehyde Dehydrogenase Type XII
(ALDH12) and Retinal Dehydrogenase Type II (RalDH2) are responsible
for the oxidation of the cis and trans forms of retinal to retinoic
acid respectively. The goal of this research was threefold:
1) to create the expression plasmids that contain the genes
of ALDH12 and RalDH2 in their native forms 2) overexpression
ALDH12 and RalDH2 and 3) preliminary purification of RalDH2
and ALDH12 for crystallization at a later date. It is important
for RalDH2 and ALDH12 to be crystallized in their wild-type
form because that would provide the most accurate and detailed
picture of their structure. Previous experiments have yielded
a crystallized configuration of RalDH2 that contained a Histidine-tag,
which may have affected its structure and contributed to its
substandard resolution. Additionally, mutant constructs of both
RalDH2 and ALDH12 were created to change their active sites,
which will hopefully allow for the substrate, retinal, to be
crystallized with the enzyme. The genes coding for the native
forms ALDH12 and RalDH2 were originally in plasmids unsuitable
for large scale expression. The genes were transferred to pET-3a,
which is a plasmid that can be used for overexpression. The
purification steps consisted of using Dye Affinity Chromatography
and washing with different concentrations of NaCl.
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