Transcript IL-13 - York College of Pennsylvania

Design and Production of a GFP and Human IL-13
Linked Chimeric Protein in E. coli Using pQE-30 Vector
Stephen R. Suknaic
Department of Biology, York College of Pennsylvania
Structure of GFP
Structure of IL-13
Introduction
Methods
Results
Conclusions
Interleukin 13 (IL-13) is an immunoregulatory cytokine that has been a
useful tool in breast and brain cancer
research (Kawakami 2004). This protein is
a ligand for the surface receptors IL13Rα1 and IL-13Rα2. These receptors are
effective targets for cancer research due
to their over-expression on the surface of
certain cancer cell types.
This project began with the design of a
protein with the following structure:
The following amino acid sequence
was inferred from the final pQE-30 DNA
sequence
obtained
from
Elim
Biopharmaceuticals:
 All the amino acid codons for GFP
IL-13 ligand has been experimentally
attached to other proteins to produce a
new fusion or ‘chimera’ protein. By
successfully developing a fused protein
consisting of more than one active part,
researchers
have
been
able
to
successfully develop a single protein with
multiple functions and potential uses. A
prime example of this is a fusion of IL-13
and Pseudomonas exotoxin A, which
can carry a toxic molecule to a cell
expressing the IL-13 receptor.
The
primary
objective
of
this
experiment is to see if IL-13 could be
successfully fused with Green Fluorescent
protein to create a green glowing
chimera protein containing the IL-13
ligand. Such a protein would have many
theoretical uses:
Competition
assays
with
targeting the IL-13Rα2 Receptor
drugs
Quantification of the number of IL-13
receptors on a cell membrane
Luminescence can be used to identify
tumor locations or sizes
His-Tag and
Entero-kinase
Cut Site
GFP
Linker Region
and Factor
Xa Cut Site
IL-13
The following timeline is a summary of the
protocols involved with this experiment:
Designed Primers for GFP and IL-13
PCR Amplified Stock DNA for GFP and
IL-13 Using Custom Primers
Digestion and Ligation of GFP DNA and
pQE-30 Vector with Restriction Enzymes
Verify Construct with DNA Sequencing
Transformation of GFP/pQE-30 Vector
into Competent E. coli
Harvested and Purified Plasmid
from Transformed E. coli
Histidine Tag
GFP
Factor Xa Cut Site
Enterokinase Cut Site
Linker Region
IL-13
 Modified
pQE-30
successfully created
produce chimera
vector
was
and used to
 The proper structure of this protein
was confirmed by protease digests, Ionaffinity chromatography, and presence
of the characteristic glow of Green
Fluorescent Protein
Take Home Message
 Histidine Tag presence and function
was verified by testing the chimera for
affinity to Nickel- ion beads using IMAC
 The GFP and IL-13 chimera was
successfully made using the pQE-30
vector as a means to transform E. coli
 Enterokinase Cut Site presence was
confirmed by treatment with Enterokinase
enzyme to release the protein from the
beads
 The components of this protein were
tested to confirm their presence
beyond just their amino acid sequence
Digested Recovered Plasmid and
IL-13 DNA with Restriction Enzymes
 GFP presence was confirmed by the
characteristic bright green glow
 Future studies could test to see if the
chimera will bind effectively to the IL-13
α1 and/or α2 receptor
Ligation of IL-13 DNA into Vector
 Linker Region presence was confirmed
by the DNA sequencing
Works Cited
Vector Transformation into
Competent E. coli
Grow to O.D. 600 ≈ 1.0 Abs
Induced Protein Production with
IPTG Treatment
Harvested E. coli and lysed
with B-per Reagent
Purified Inclusion Body and Isolated
Chimera via Ion-Affinity Chromatography
Primary Objectives
I. Integrate modified DNA sequences for
GFP and IL-13 into pQE-30 vector
 Factor Xa Cut Site presence was
confirmed by treatment of the chimera
with Factor Xa enzyme
 IL-13 presence was supported by the
polyacrylamide gel run after the Factor
Xa enzyme treatment
Figure 1: Structure of the Quiagen pQE-30 Vector
GFP and
Linker ~30 kd
Intact
Chimera
~42 kD
kD
50
37
25
20
Factor Xa
enzyme
15
10
IL-13 ~12 kd
1
II. Transform said vector into competent
E. coli and induce protein production
with IPTG
III. Isolate chimera and analyze it for
proper biological activity
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and IL-13 were properly integrated in
sequence into the pQE-30 vector
2
3
4
Figure 2: Polyacrylamide gel showing the intact chimera as
well as after digestion with Factor Xa enzyme. The IL-13 that
was digested is running at ~12 kD. Lane 1 contained standard
kaleidoscope ladder. Lane 2 contained only the purified
chimera with a mass of ~42kD. Lane 3 contained the chimera
digested with Factor Xa enzyme. Lane 4 contained only the
Factor Xa enzyme.
Kawakami K, Kawakami M, Puri RK. Specifically targeted
killing of interleukin-13 (IL-13) receptor-expressing breast
cancer by IL-13 fusion cytotoxin in animal model of human
disease. Molecular Cancer Theory. 2004 Feb;3(2):137-47.
Kawakami K, Kawakami M, Puri RK. IL-13 receptortargeted cytotoxin cancer therapy leads to complete
eradication of tumors with the aid of phagocytic cells in
nude mice model of human cancer. Journal of
Immuonology. 2002 Dec 15;169(12):7119-26
http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=S
howDetailView&TermToSearch=3596
http://www.conncoll.edu/ccacad/zimmer/GFPww/structure.html
http://www.rcsb.org/pdb/explore/images.do?structureId=1I
JZ
http://www1.qiagen.com/Products/Protein/Expression/QIAe
xpressExpressionSystem/pQE-30XaVector.aspx?ShowInfo=1
Acknowledgements
I would like to thank Dr. Thompson for his guidance,
wisdom and support throughout the duration of this project. I
would also like to thank the entire York College Biology
Faculty Staff for any help or suggestions that they have
made with regards to this project.