Production of the PDZ TIAM 1 QM Protein

Kaitlyn Kutsch, David Speckhard Ph.D.

Loras College Department of Biochemistry, Dubuque, IA

INTRODUCTION
TIAM stands for tumor induction and metastasis and
has been found in many human cancer cells. The PDZ
domain is heavily involved and important in signal
transduction. The PDZ domain is very important in
signaling for cell movement and specifically
metastasis. The thing that is believed to be crucial in
sending these signals is the PDZ domain binding
pocket. This binding pocket sends internal signals to
the cell that controls messenger proteins. PDZ domains
bind to a short region of the C-terminus of other
specific proteins. When this terminus enters the PDZ
binding pocket it triggers a specific signal. My project
was to work with the PDZ QM protein. QM stands for
quaternary mutant. The QM protein is one that is not
found in humans, rather is was made in a lab. It is an
intermediate blend between TIAM 1 and TIAM 2. This
protein is not naturally occurring, but it can be very
important to help understand at a basic level how
structure changes function.

RESULTS
The PET30 plasmid was successfully cut and
the YFP fragment was obtained using an
agarose electrophoresis.

2.Long term goals of our study is

to find what binds to the PDZ
domain most tightly and develop
a drug that mocks this binding
and stops the spread of cancer.

Some problems I ran into were that the

ligase was not working. It was
discovered that the ligase was expired
and inactive. I also ran into issues with
my ampicillin plates growing everything.

FUTURE WORK
This figure shows the YFP fragment in the 6 th lane at 750 base pairs.

This isolated fragment was run through a

PCR. The PCR successfully amplified this
DNA fragment and used specific primers to
add different restriction sites needed for
ligation.

STUDY AIMS
1.Modify DNA to express the QM
protein with YFP (yellow
fluorescent protein) to define the
specificity and function of the QM
PDZ domain.

LIMITATIONS

This figure shows the amplification of YFP with new restriction sites.

The PET 21a plasmid containing the

sequence for the PDZ QM protein was
successfully isolated from DE3 cells. This
plasmid was then successfully cut using a
NCO1 nuclease.

In the future I will use a new ligase to

link the YFP fragment and DE3 DNA
plasmid. Once this plasmid has been
successfully ligated, I will isolate and
inoculate the glowing cells. These cells
will be inoculated and grown up to
express the PDZ QM protein with the YFP
fragment. I will induce the cells with
IPTG in order to produce a large number
of the PDZ QM protein. Once this protein
is isolated and expressed, peptides
known to bind with the PDZ domain will
be introduced and the strength of
binding will be tested. The strength of
this binding will be tested using a
technique call FRET (Fluorescence
Resonance Energy Transfer). FRET will
be achieved by using a BFP(blue
fluorescent protein.

METHOD
1. A double restriction digest was run to
cut out the YFP protein from PET 30
plasmid.
2. PCR was run on the YFP fragment to
amplify sample and add correct
restriction sites
3. The DNA from the PDZ QM protein was
isolated from DE3 cells
4. The DE3 cells were isolated and cut
using restriction digest to allow the
YFP fragment to be pasted in
5. The DE3 plasmid with YFP was
transformed and ligase was added to
connect the plasmid and YFP vector.

LORAS.EDU

A shows the isolation of the PDZ QM protein plasmid. This is the

plasmid as characterized by the three bands present in the third
lane. B shows the cut DE3 DNA in the 5th lane. This cut DE3
collapses into one band indicating it has successfully been cut.

The ligation transformation was

unsuccessful as no colonies grew on an
ampicillin plate.

REFERENCES
Shepherd, T. R., and Fuentes, E. J. (2011) Structural and
thermodynamic analysis of PDZ-ligand interactions, Methods
Enzymol 488, 81-100.
Shepherd, T. R., Hard, R. L., Murray, A. M., Pei, D., and
Fuentes, E. J. (2011) Distinct ligand specificity of the tiam1
and tiam2 PDZ domains, Biochemistry 50, 1296-1308.