Centennial Honors College
Western Illinois University
Undergraduate Research Day 2012
Poster presentation
Expression of Human L-3-hydroxyacyl-CoA Dehydrogenase
Kelly Delehanty, Matthew Crawford, and Sam Wrobel
Faculty Mentors: Jenq-Kuen Huang and Lisa Wen
Chemistry
L-3-hydroxyacyl-CoA Dehydrogenase (L-3-HAD) is one of the enzymes involved in
fatty acid β-oxidation pathway, a process in which fatty acids are broken down and
release energy. Our interest in obtaining a functional recombinant L-3-HAD is to use
it as a positive control in our ongoing research.
Previously, a novel secondary alcohol dehydrogenase (2o-ADH) was cloned from
Micrococcus luteus WIUJH20 (M. luteus WIUJH20). It was discovered that the
enzyme belonging to the L-3-HAD family based on amino acid sequences alignment
with other homologous proteins from the Protein Database. However, unlike L-3HAD family the 2o-ADH has broad substrate specificity. To compare substrate
specificities between 2o-ADH and L-3-HAD, it is necessary to clone and express L-3HAD from M. luteus WIUJH20. The cloning and expression of an annotated L-3-HAD
from M. luteus WIUJH20 has recently been completed in this lab. However, the
recombinant protein has no detectable activity which could be due to a number of
possible reasons. To see if our activity assay protocol was valid, we need a known
active L-3-HAD (i.e. Human L-3-HAD) to serve as a positive control. Human L-3HAD gene was amplified by PCR. The amplified gene was subcloned into pET28a
vector via EcoRI site. This was followed by transformation into JM109, and then
BL21(DE3)pLysS host cells. The human recombinant protein was successfully
expressed after 4 hours of ITPG induction in both LB and 2xYT cultured media.
Currently, the enzyme is being analyzed for activity.