Protein kinases are among the most important proteins in signal transduction in
mammalian cells. There are more than 500 different human protein kinases. In this work,
we focused on delta isotype of protein kinase C (PKCδ), which is involved in regulation
of many cell processes, such as apoptosis, differentiation and proliferation. It is a 78 kDa
protein, composed of N-terminal regulatory region and C-terminal catalytic region. The
structure of PKCδ catalytic region has not been determined yet. It has been shown that
the cytoplasmic tail of epithelial cell adhesion molecule (EpCAM) interacts with PKCδ
catalytic region and therefore inhibits it. We wanted to prepare PKCδ for the purpose of
further research on PKCδ-EpCAM interaction.
We have expressed PKCδ catalytic region in bacterial cells, yet we did not acquire
adequate amount of soluble recombinant protein. Using the baculovirus expression
system, the expression of PKCδ catalytic region and full length protein was carried out in
insect cells. We have successfully expressed full-length PKCδ, which we identified with
mass spectrometry. However, we could not remove impurities from the sample using
affinity chromatography and ion exchange chromatography. Using molecular cloning
techniques, we have also prepared constructs of catalytic region and full-length protein in
fusion with fluorescent protein mCherry for the purpose of further investigation of PKCδ.
Our results serve as a basis for further research or optimization of PKCδ preparation,
while fusion constructs can provide novel tools for research of mechanisms of PKCδ action
and intracellular signalling in vivo and in vitro.
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