Transcript The addition of SP-HMM34 peptide secretion signal to IL13Rα2

A Study of a Peptide Secretion Signal Addition to
Human Interleukin 13 Receptor Alpha 2 and Green Fluorescent Protein
Jacqueline Freebery and Jeffrey P. Thompson, Ph.D.
http://cancerres.aacrjournals.org/content/vol64/issue24/cover.shtml
INTRODUCTION
•Eukaryotic cells transcribe their DNA into mRNA in the
nucleus; the mRNA translation to amino acids is accomplished
in the cytoplasm at the ribosome.
Department of Biology,York College of Pennsylvania,York, PA 17405
METHODS
http://expasy.org/spotlight/back_issues/2001/06/the_greenest_of.shtmlGFP
DISCUSSION
RESULTS
HMM Secretion Sequence
Selection
1
2
3
l
•The Ligase Chain Reaction was successful in creating
the primer coding for the secretion signal.
•All four plasmids were successfully created.
•Protein trafficking is determined in some eukaryotes by
peptide signals contained within proteins that target their end
locations. The majority of secreted proteins are secreted by a
secretory pathway that is directed by secretory signal peptides
(Barash et. al. 2002).
•A Hidden Markov Model (HMM) has been computer
generated to determine artificial signal peptide sequences; using
known human signal peptides as a training set, it functions to
predict, identify, and generate signal peptides that direct strong
protein secretion and expression (Barash et. al. 2002).
•Human Interleukin 13 Receptor Alpha 2 (IL13Rα2) is
significant in tumor biology because it binds and internalizes
the Interleukin 13 Ligand with higher affinity than the IL13Rα1
without mediating signal transduction (Kawakami et. al. 2003).
• Green Fluorescent Protein (GFP) is a useful tool for protein
targeting in living cells. Engineering of GFP into chimeric
proteins is valuable in biochemical research.
OBJECTIVES
• Obtain IL13Rα2 in a secretable form so that it is
manufactured by the cell and emitted into surrounding media
instead of staying attached to the cell membrane or trapped
in the cytoplasm.
Primer Design
•Initial transfections of U-87MG cells were successful.
•Cell death occurred in U-87MG transfectants with
plasmids containing GFP containing.
LCR
•U-87MG transfectants with IL13Rα2-sec produce
IL13Rα2.
PCR
Figure 2. Plasmid used to create
proteins containing SP-HMM34
Plasmid
Restriction
Digestion
10
Ligation Reaction
9
8
7
6
5
Figure 1. SP-HMM34 peptide sequence and back translation for
encoding cDNA sequence
2
3
4
5
6
7
8
9
10
•Molecular weights of all positive results in the
comparative western blot are heavier than the weight of
the protein portion of IL13Rα2, indicating that posttranslational events have occurred.
Plasmid Purification
CONCLUSION
Nucleotide Sequencing
•The addition of the SP-HMM34 peptide secretion
signal to IL13Rα2 does not enhance secretion into
surrounding media. Therefore, the initial hypothesis,
H1, is rejected, and H0 is accepted.
Stable Transfection of
U-87MG Cell Line
Figure 4. Western Blot Analysis
showing positive results for positive
control (6), and IL13Rα2-sec
transfectant media (8) and cell pellet
(10). Negative results are shown for
blasticidin resistant transfect media (7)
and cell pellet (9).
HYPOTHESES
H0: The addition of SP-HMM34 peptide secretion signal to
IL13Rα2 and GFP will not enhance their secretion into
their surrounding media.
1
•However, the CRP10 cell line produces and secretes a
greater amount of IL13Rα2.
Transformation in E. coli
•Determine whether the addition of SP-HMM34 peptide
secretion sequence to IL13Rα2 and GFP will enhance their
secretion into the media.
Plasmid with Plasmid with Plasmid with
Empty
Plasmid
IL13Rα2-sec GFP-sec
GFP alone
H1: The addition of SP-HMM34 peptide secretion signal to
IL13Rα2 and GFP will enhance their secretion into their
surrounding media.
•U-87MG transfectants with IL13Rα2-sec secrete
IL13Rα2.
Figure 3. Gel Electrophoresis
showing 100 BP ladder (1) and 500
BP Ladder (2), followed by plasmid
digestion with endonucleases AgeI
and BspHI to show SPHMM34
secretion fragment (3).
Figure 5. Western Blot Analysis showing positive
results for positive control (2), IL13Rα2-sec cell
pellet (6), CRP5 cell pellet (8), CRP10 media (9),
and CRP10 cell pellet (10). Negative results are
shown for blasticidin resistant media (3) and cell
pellet (4), IL13Rα2-sec media (5), and CRP5 media
(7).
Media and Cell
Analysis
Affinity Chromatography
Sample
Migration Distance
(mm)
LOG of the
Molecular Weight
Molecular Weight
(kD)
IL13Rα2-sec Cell Pellet
CRP5 Cell Pellet
CRP10 Media
CRP10 Cell Pellet
5.3
4.8
4.7
4.8
1.60
1.70
1.71
1.70
40.01
49.67
51.86
49.67
Figure 7. Shows data for measured migration
distances (mm) of positive results from comparative
western blot along with molecular weight
calculations from linear regression equation.
SDS-PAGE
Western Blot Analysis
•The effect of the SP-HMM34 peptide secretion signal
to GFP is unknown due to the unforeseeable occurrence
of cell death in the U-87MG transfectants with GFP. A
repeat study with the addition of SP-HMM34 to GFP is
needed to determine a definite conclusion.
LITERATURE CITED
Barash, S., Wang, W., Shi, Y. Human secretory signal peptide description by hidden
Markov model and generation of a strong artificial peptide for secreted protein
expression. Biochemical and Biophysical Research Communications 2002. 294:
835-842.
Kahlon, K.S., Brown, C., Cooper, L.J., Raubitschek, A., Forman, S.J., and Jensen,
M.C. 2004. Specific recognition and killing of glioblastoma multiforme by
interleukin 13-zetakine redirected cytolytic T cells. Cancer Res. 24:9160-9166.
Kawakami, K., Kawakami, M., Husain, S.R., Puri, R.K. 2003. Potent antitumor
activity of IL-13 cytotoxin in human pancreatic tumors engineered to express IL13 receptor 2 chain in vivo. Gene therapy [serial online] 10:1116-1128. Available
from: http://www.nature.com/gt/journal/v10/n13/full/3301956a.html
Tsien, R.Y. 1998. The Green Fluorescent Protein. Annual Review of Biochemistry.
67: 509-544.
ACKNOWLEDGEMENTS
MRPTWAWWLFLVLLLALWAPARG
ATG CGC CCC ACC TGG GCC TGG TGG CTG TTC CGT GTG
CTG CTG CTG GCC CTG TGG GCC CCC GCC CGC GGC
Figure 6. Shows standard curve to
determine the molecular weights of
standards.
I would like to thank Dr. Thompson for his continued guidance and support
throughout this study.