Cloning and Sequencing of Point Mutants of GluT1
Christine Ikponmwonba and Eric Arnoys
Department of Chemistry & Biochemistry, Calvin College, Grand Rapids, Michigan 49546
Introduction
Glut 1 is one of the isoforms of glucose
transporters. It is a membrane bound
transporter found in most eukaryotes. Its
role in glucose metabolism is to contribute
mainly to basal uptake of glucose. Because
of the central role of glucose in metabolism,
drugs that act on Glut1 might be used to
treat diabetes and cancer. Monitoring the
activity of Glut1 protein through mutagenesis
and cloning can lead to new discoveries.
Results
Objectives
Investigate the importance of each of the six
cysteine residues in Glut 1 by site directed
mutagenesis and express them in life cells
3. PCR fragments were inserted into a shuttling
vector using TOPO cloning, and the resulting
vector was transformed into E. coli with heat
shock.
We also successfully created new restriction sites
and expressed the Glut1 mutants as GFP fusion
proteins.
Figure 5.
Mutant Glut 1
with new
restrictions
sites
Methods
Figure 1. Model Glut 1
protein with cysteine
groups embedded in the
cell membrane
We successfully replaced half of the cysteine
residues with serine through site-directed
mutagenesis.
We performed site-directed mutagenesis on Glut 1,
substituting the cysteine residues for each serine.
4. The DNA was purified and then digested with
restriction enzymes specific for the newly
inserted sites.
5. QlAquick Gel Extraction Kit Protocol to extract
the DNA fragments with new restriction sites.
6. Recovered DNA was then ligated into GFP
(an expression vector) and then transformed.
Figure 3.
Sequence with highlighted substitution
Final product was stored for sequencing.
Purified DNA contained the inserted Glut 1 mutants.
From this, we will monitor closely the mode of
Glut1 activation in GFP.
Presently,
More we are working on two prosposed
biological pathways, oligomerization and Lipid raft
association, through FRAP experiments to also
see how Glut 1 is activated.
Future Work
More research is still being done on our proposed
mechanism that disulfide bonds help in the activation
of Glut 1.
We hope to perform FRAP and activity experiments
on the Glut 1 mutants to determine if they can be
activated.
Also, more of the FRAP experiments will help us
determine if oligomerization occurs.
Experents
From past research on Glut 1, we
have proposed that Glut 1 is
activated by the formation of
internal disulfide bonds.
1. We used Polymerase Chain Reaction (PCR) to
produce millions of copies of the desired DNA
sequence. At the same time, the PCR adds new
restriction sites to the end of the DNA piece.
2. Agarose Gel Electrophoresis to separate the DNA
fragments which contained about 20ng of DNA
Bands were views under high intense UV light
Figure 2.
Glut 1 proposed mechanism
GLUT1 is thought to be activated by
the formation of internal disulfide
bond and subsequent tetramer
formation. When the thiol (-S-X)
within GLUT1 has been modified,
there are three possible effects that
identify changes in GLUT1 structure
• Inhibition of glucose uptake
• Activation of transport
• Inhibits agents from activating
basal uptake.
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Ladder
Figure 4.
Agarose gel showing
1500bp band of mutants
Time (s)
Figure 6.
Before and
after pictures
of Glut 1
mutant
expressed in
GFP
Acknowledgments
I want to thank Professor Eric Arnoys for his
mentorship, Professor Larry Louters for his
professional assistance and for their
previous work on this project. I also want to
thank the other members of the Louters and
Arnoys labs: Elizabeth De Groot, Riemer
Praamsma, Justice Mason, Ola Alabi, Steven
Gunnink and Ben Kuiper for their general
assistance.
This project was funded by the National
Institutes of Health.