به نام مهربانتر ین in the name of the most compassionate 1
TRANSCRIPT
![Page 1: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/1.jpg)
1
به نام مهربانت In the nameرین
of the most compassiona
te
![Page 2: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/2.jpg)
2
Design and Construction of Antagonistic VEGF Variant for Inhibition of Angiogenesis
By:Fahimeh Ghavamipour
Supervisors:Dr. R. H. Sajedi
Dr. M. Aghamaali
Advisors: Dr. S. S. Arab
Dr. K. Mansouri
Dept. of Biology, Faculty of Sciences, University of Guilan
![Page 3: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/3.jpg)
Cancer
• The division of normal cells is precisely controlled. New cells are only formed for growth or to replace dead ones.
• Cancerous cells divide repeatedly out of control even though they are not needed, they crowd out other normal cells and function abnormally. They can also destroy the correct functioning of major organs.
3
![Page 4: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/4.jpg)
4
Tumor Growth
• Phase 1: Genetic mutations– Cellular cycle and apoptosis disruption– Uncontrolled reproduction, no cell death
• Phase 2: Interaction with immune system – Cancer cells inhibit immune system
• Phase 3: Solid tumor– Cancer cell diffusion– Necrotic zones– Solid tumor diameter 1-2 mm
Necrotic zone
Uncontrolled Reproduction
Healthy cells
![Page 5: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/5.jpg)
5
Angiogenesis and Metastasis
• Tumor growth requires nutrients– Active nutrient search
• Phase 4: Angiogenesis– Segregation of proteins which promote
blood vessel growth– Aberrant vascular network
• Phase 5: Metastasis– Cancer cells enter in blood network– New colonies in healthy regions
![Page 6: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/6.jpg)
6
Angiogenesis
Angiogenesis :
Sprouting of new tubes
off of pre-existing tubes
![Page 7: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/7.jpg)
7
The Angiogenesis Signaling Cascade
Genes are activated in cell nucleus
Cancer cell
VEGF (or bFGF)
Endothelial cell surfaceRelay
proteins
Receptor protein
Proteins stimulate new endothelial cell growth
![Page 8: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/8.jpg)
8
Activators of Angiogenesis
Tumor Angiogenic Factors (TAF)
VEGF : vascular endothelial growth factor
bFGF: basic fibroblast growth factor
![Page 9: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/9.jpg)
9
VEGF & Angiogenesis
•VEGF or VEGFA is the most potent & dominant stimulator of angiogenesis
• A key factor in tumor growth, progression and metastasis
•Highly expressed in several tumor types
•4 isoforms:VEGF121, 165, 189, 206
![Page 10: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/10.jpg)
10
Multiple Isoforms of VEGF-A are Generated from Exon Splicing
Highly diffusible isoform
1 121VEGF-A121
1 206
Highest molecular weight isoform bound to extracellular matrix
VEGFR-bindingdomain
Heparin-bindingdomain
VEGF-A206
Sequestered in the extracellular matrix
1 189VEGF-A189
1651
Most abundant isoform expressed in humans
VEGF-A165
VEGF8-109 is a segment from VEGF-A
![Page 11: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/11.jpg)
11
• Homodimeric glycoproteinis• Held together by two intermolecular disulfide bonds• MW 24 KD• Have receptor binding domains in each monomer
VEGF8-109
![Page 12: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/12.jpg)
12
VEGF and VEGF Receptors
![Page 13: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/13.jpg)
13
VEGF Receptors
VEGFR I, Flt-1Higher affinity to VEGFAct primarily as a decoy receptorModulating the availability of VEGF to VEGFRII
VEGFR II, KDRLower affinity to VEGFThe principal receptor for VEGF signaling
This mutational analysis implicates KDR, but not Flt-1, has a critical role in VEGF
induction of endothelial cell proliferation
![Page 14: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/14.jpg)
14
KDR Mechanism of Action
video.mp4
oDimeric structure of VEGF is a prerequisite of receptor activation
oOne VEGF molecule bridges two receptors via two similar recognition sites
oVEGFR-2 induces angiogenesis through activation of the classical extracellular regulated kinase pathway, leading to signal transduction
![Page 15: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/15.jpg)
15
Inhibitors of
o Growth factorso Endothelial cell signal transductiono Endothelial cell proliferationo Endothelial cell migrationo Matrix metalloproteinaseo Endothelial cell survivalo Bone marrow precursor cells
Given its central role in promoting many cancers, VEGF provides an attractive target for therapeutic intervention
Angiogenesis inhibitors
![Page 16: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/16.jpg)
16
Strategies for VEGF Inhibition
• Targeting the ligand (e.g. VEGF antibodies such as Avastin)
• Targeting the receptor (e.g. VEGF antagonists)
Video
![Page 17: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/17.jpg)
17
The Aim of Study
Construction of a heterodimeric antagonistic VEGF variant (HD-VEGF)
• Binding domains for the VEGF-receptor KDR/Flk-1 is present at one pole of the dimer, whereas the other pole carries domain swap mutations
• HD-VEGF blocks KDR dimerization and KDR-mediated VEGF activities that are crucial in the angiogenic process
VEGFl3165/VEGFl2
121 (William Leenders, et al.)
VEGFl1/VEGFl2 (Germaine Fuh, et al.)
Hv1 (Niv Papo, et al.)
scVEGF (Gerhard Siemeister, et al.)
Problem of previous studies:PurificationHighe IC50
![Page 18: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/18.jpg)
18
In this study
Theoretical study
- Mutation Design
- Modeling and MD Simulation
- Molecular Docking
HD-VEGF construction
- Gene Synthesis
- Expression, Refolding & Purification of heterodimeric VEGF
Structural & Functional Studies
- CD & Fluorescence Analysis
- HuVEC Proliferation & Capillary Tube Formation Assay
![Page 19: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/19.jpg)
19
Variation of the mean accessible surface areas, ∆ASA (Å2), with the amino acid residues number for VEGF RBD (PDB ID: 3V2A)
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 1150
20
40
60
80
100
120
140
Residues Number
∆ A
SA
(Å
)Identification of KDR Binding Site by Using
ASA Analysis
Theoretical study
(Brozzo, M.S., et al., Blood, 2012. 119(7): p. 1781-1788).
![Page 20: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/20.jpg)
20
Variation of the mean accessible surface areas, ∆ASA (Å2), with the amino acid residues number for VEGF RBD (PDB ID: 2VPF)
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 1150
20
40
60
80
100
120
140
Residues Number
∆ A
SA
(Å
)
Theoretical study
Identification of Amino Acids Involved in dimerization by Using ASA Analysis
![Page 21: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/21.jpg)
21
0 20 40 60 80 100 1200
20
40
60
80
100
120
140
2VPF
3V2A
Residues nmmber
∆A
SA
Å2
Theoretical study
![Page 22: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/22.jpg)
22
Identification of KDR Binding Domains
By using crystal structure of :
VEGF RBD (PDB ID: 2VPF)
VEGF-KDR complex (PDB ID: 3V2A)
Alanine scanning studies
The amino acids involved in KDR binding and protein dimerization were analyzed
Theoretical study
![Page 23: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/23.jpg)
23
Theoretical study
Segment 41-46 Segment 81-87
Mutant VEGFG Q N H H E V V K F M D V Y Q R S Y C H P I E T L V D I F Q E Y P G E G D E I L T F K P S C V P L M R C G G C C N D E G L E C V P T E E S N I T M Q I H G L R P S V A G Q H I G E M S F L Q H N K C E C R P K K D
Wild type VEGF G Q N H H E V V K F M D V Y Q R S Y C H P I E T L V D I F Q E Y P D
E I E Y I F K P S C V P L M R C G G C C N D E G L E C V P T E E S N I T M Q I M R I K P H Q G Q H I G E M S F L Q H N K C E C R P K K D
Mutation Design
![Page 24: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/24.jpg)
24
Theoretical studyTheoretical study
12Å 13Å
Selected Segments for Mutation
• These suitable sequences were searched in
Protein Data Bank with some criteria such as:
•Having similar dihedral angles•The same distance between amino and carboxy terminals in segments•Their conformations
![Page 25: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/25.jpg)
25
Modeling
• A 3D structure model of mutant VEGF was constructed based on wild type VEGF (PDB ID:2VPF) structure by using the I-TASSER program
• I-TASSER is best program in casp7 & casp8
• The model of designed mutant VEGF was compared to native one after energy minimization process
Theoretical study
Zhang, Y. BMC bioinformatics, 2008. 9(1): p. 40Roy, A., A. Nature protocols, 2010. 5(4): p. 725-738.Roy, A., J. Yang, and Y. Zhang, Nucleic Acids Research, 2012. 40(W1): p. W471-W477.
![Page 26: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/26.jpg)
26
Molecular Dynamic Simulation
The selected model of mutant VEGF was used as initial structures for MD.
MD was done by GROMACS software Minimization was done during 5000 steps The simulation was performed in 300 °K GROMOS43a1 force field was used The simulation was run with 2 fs Time steps The explicit model H2O with 8 Å thickness was used for the
simulation The simulation was run for 1 ns
Theoretical study
![Page 27: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/27.jpg)
27
Stability Investigations for MD Simulation
RMSD was stabilized nearly around 100 ps (MD production)
Total energy was stabilized nearly around 10 ps (Equilibration phase)
0 100 200 300 400 500 600 700 800 900 1000
-0.0200000000000001
-4.16333634234434E-17
0.02
0.04
0.06
0.08
0.1
Time(ps)
RM
SD(n
m)
1 11 21 31 41 51 61 71 81 91
-245000
-243000
-241000
-239000
-237000
-235000
Time (ps)
Tot
al e
nerg
y (K
cal/
mol
)
0 10 20 30 40 50 60 70 80 90 10050000
52000
54000
56000
58000
60000
-300000
-298000
-296000
-294000
-292000
-290000
Kinetic EnergyLinear (Kinetic Energy)
Time (ps)
K-e
nerg
y (K
cal/
mol
)
P-e
nerg
y (K
cal/
mol
)
Theoretical study
![Page 28: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/28.jpg)
28
Temperature & Density was stabilized at 300 oK & 1.02 gr/cm3 (Equilibration phase)
Radius of gyration was stabilized
Theoretical study
Stability Investigations for MD Simulation
0 200 400 600 800 1000250
300
350
400
Time (ps)
Tem
pera
ture
(K)
0 200 400 600 800 1000990
1000
1010
1020
1030
1040
Time (ps)
Den
sity
(kg/
m3)
0 100 200 300 400 500 600 700 800 900 10001.78200000000001
1.78500000000001
1.78800000000001
1.79100000000001
1.79400000000001
1.79700000000001
1.80000000000001
Time (ps)
Rad
ius o
f Gyr
atio
n (n
m)
![Page 29: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/29.jpg)
29
Molecular Docking
• The proposed structural model of HD-VEGF and KDR crystal structure
were used for docking
• Docked molecular complexes were constructed using 3D-Dock program
• 10000 complex were suggested by 3D-Dock as the best complexes
Theoretical study
![Page 30: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/30.jpg)
30
Conclusion I
Identification of KDR binding domain was performed and replace by
suitable segments
A 3D structure model of mutant VEGF was constructed successfully
The model was refined through MD simulation
MD simulation showed good stability in Stability Investigations
Study on the docked complexes in compare with native complex shows
having incorrect contact or wrong orientation between complex of
VEGF mutant model and KDR.
Theoretical study
![Page 31: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/31.jpg)
31
Gene Synthesis & Transformation
• The receptor binding domain of mutant VEGF gene was designed, synthesized and inserted in pET-21a+ expression vector
• Via NheI and BamHI sites with additional N-Terminal Strep tag
• Plasmid was transformed into the competent cells of E. coli (BL21) expression system. using Sambrook chemical method
HD-VEGF construction
![Page 32: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/32.jpg)
32
Sequencing
Plasmid extraction and sequencing was
used to confirm the fidelity of the amplified
fragment
HD-VEGF construction
![Page 33: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/33.jpg)
33
Expression Of Wild type & Mutant VEGF
• Inducing of positive colony by 1mM IPTG & 2mM lactose in 27 oC
• Monitoring of wild type & mutant VEGF expression using reducing
SDS-PAGE analysis
• The receptor binding domain of Native & mutant VEGF was expressed
as inclusion body in E. coli
HD-VEGF construction
![Page 34: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/34.jpg)
34
To production of heterodimeric VEGF
The mutant and Native VEGF were mixed in an approximately equimolar ratio, and the refolding was performed on the mixture through multi-step refolding procedure
After the refolding the solution was expected to contain [1/4 NativeVEGF homodimer (N-VEGF) , 1/2 heterodimeric VEGF (HD-VEGF), 1/4 mutant VEGF homodimer (M-VEGF) ]
N-VEGF HD-VEGF M-VEGF
RefoldingHD-VEGF construction
![Page 35: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/35.jpg)
35
Reducing & Non-reducing SDS-PAGE
Considering to the fact that VEGF dimerization is essential for its
receptor binding and biological activity, we followed dimerization of
the protein in the refolding process using reducing and non-reducing
SDS-PAGE.
Refolded protein was present primarily in the dimeric form with little
amount of monomer
HD-VEGF construction
Reducing Non reducing
![Page 36: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/36.jpg)
36
H
H
S S
SH
N-VEGF HD-VEGF M-VEGF
Purification By Two Step Affinity Chromatography System
HD-VEGF construction
His tagged & Strep tagged heterodimeric variant
![Page 37: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/37.jpg)
37
Purification By Two Step Affinity Chromatography System
0 2 4 6 8 10 12 14 16 18 200
0.2
0.4
0.6
0.8
1
0
50
100
150
200
250
300
Volume (ml)A
280
Imid
azol
e (m
M)
0 1 2 3 4 5 6 7 8 9 100
0.2
0.4
0.6
0.8
1
0
0.5
1
1.5
2
2.5
3
Volume (ml)
A28
0
Des
tiob
ioti
n (
mM
)
HD-VEGF construction
Purification of His-tagged and Strep-tagged heterodimeric protein was carried out using Ni-NTA Agarose & Strep-Tactin column
![Page 38: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/38.jpg)
38
Strep- TactinHD-VEGF construction
![Page 39: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/39.jpg)
39
Purified HD-VEGF
Non-reducing SDS-PAGE showed a single 28 kDa band indicating that the protein was purified to homogeneity
HD-VEGF construction
Gerhard Siemeister, et al. 1998.
![Page 40: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/40.jpg)
40
Conclusion II
Sequencing confirmed the construction of mutants VEGF
The mutant VEGF was expressed as inclusion bodies successfully.
The mutation had no effect on protein dimerization.
Native and mutant VEGF were successfully refolded and dimerized. Also the heterodimeric variant was obtained in dimeric form.
Purified HD-VEGF variant was obtain whit very good quality by using two step affinity chromatography system.
HD-VEGF construction
![Page 41: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/41.jpg)
41
As we aspect, the Far-UV CD spectra of M-VEGF, HD-VEGF & N-VEGF Shows that HD-VEGF forms are nearly similar to that of N-VEGF
M-VEGF HD-VEGF N-VEGF
12% 12% 12% Helix52% 53.5% 55% Sheet36% 34.5% 33% Random coil
Structural Analysis
Far-UV CD
190 200 210 220 230 240
-20000
0
20000
Native homodimer
Mutannt homodimer
Heterodimer
wavelength (nm)
[Ɵ](
deg.
cm2.
dmol
-1)
![Page 42: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/42.jpg)
42
Fluorescence spectroscopy
Intrinsic fluorescence
400 420 440 460 480 500 520 540 560 580 6000
20
40
60
80
100
120
Native homodimer Mutant homodimer
Heterodimer
Wavelength (nm)
Flo
ures
cnce
Int
ensi
ty
0 0.05 0.1 0.15 0.2 0.250
1
2
3
4
5
6
7
8
Mutant heterodimerLinear (Mutant heterodimer)Native homodimerLinear (Native homodimer)
[Acrylamid] (M)
f0/f
Extrinsic fluorescence Quenching fluorescence
No significant structural changes in the HD-VEGF in compare with native and mutant homodimer.Correct structure of HD-VEGF
Structural Analysis
295 315 335 355 375 3950
50
100
150
200
250
300
350
Mutant homodimer Heterodimer
Native homodimer
Wavelength
Flu
ores
cenc
e In
tens
ity
![Page 43: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/43.jpg)
43
HuVEC Proliferation Assay
M-VEGF HD-VEGF
373±3 33 2± IC50 (ng/ml)
Functional study
HD-VEGF M-VEGF
![Page 44: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/44.jpg)
44
HuVEC Capillary Tube Formation Assay
M-VEGF HD-VEGF
3±329 1±24 IC50 (ng/ml)
Functional study
Control 60 ng/ml HD-VEGF
HD-VEGF
M-VEGF
80 ng/ml HD-VEGF 480 ng/ml M-VEGF
![Page 45: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/45.jpg)
45
Previous studies
The privilege of our HD-VEGF
• Using of two step affinity chromatography system• Precise determination of amino acids involved in receptor binding• Deliberate design considering to the less effect on protein structure
and dimerization
Functional study
IC50 ng/mlRefrences Tub formation assay Proliferation assay
24 33 H-VEGF 8-109
Gerhard Siemeister, et al. 1998 - 150 VEGFl3165/VEGFl2
121
William Leenders, et al. 2002 - 200 VEGFl1/VEGFl2
Germaine Fuh, et al.1998 . - 550 Hv1
Niv Papo, et al. 2011. 227 - scVEGF
![Page 46: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/46.jpg)
46
Conclusion III
CD analysis showed secondary structural similarity between HD-VEGF and N-
VEGF protein and it was in agreement with the theoretical study.
Fluorescence spectroscopy showed No significant structural changes in the
HD-VEGF variant in compare with native and mutant homodimer, and it was
in agreement with the results of CD analysis
Investigation of anti-angiogenic properties of heterodimeric variant revealed
that this variant can significantly inhibit the proliferation and capillary tube
formation of endothelial cells in vitro
Moreover this variant possesses much higher inhibitory effect than other
VEGF antagonists which have been so far constructed
Purified HD-VEGF variant was obtain whit very good quality by using two
step affinity chromatography system.
Structural & Functional study
![Page 47: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/47.jpg)
47
Future works
• The mutation of Flt-1 receptor binding sites in order to decrease the IC50 value
• the mutation in KDR binding site in another monomer.• The precise design to modify the receptor binding domain of HD-VEGF in
order to induce higher binding affinity and subsequently lower IC50• Investigation of heterodimeric variant interaction with KDR receptor using
some techniques such as ELISA, SPR and spectroscopic methods• Supplementary anti-angiogenic and anti-tumor tests• Animal trials and in-vivo studies to investigate the anti-angiogenic and anti-
tumor effect of HD-VEGF
![Page 48: به نام مهربانتر ین In the name of the most compassionate 1](https://reader036.vdocument.in/reader036/viewer/2022070415/56649e205503460f94b0c9b4/html5/thumbnails/48.jpg)
48Thank you