Ekta Sharma, Min Dai, Jin K. Montclare* Engineering Di-Block Co-Polymers for Drug Delivery Polytechnic Institute of NYU Department of Chemical and Biological Sciences 6 Metrotech Center, Brooklyn, NY 11201 Sigma Xi Student Reserach Showcase 1
1. Engineering Di-Block Co-Polymers for Drug Delivery Ekta
Sharma, Min Dai, Jin K. Montclare* Polytechnic Institute of
NYUDepartment of Chemical and Biological Sciences 6 Metrotech
Center, Brooklyn, NY 11201Sigma Xi Student Reserach Showcase 1
2. Hypothesis: Generate Smart Biomaterials GNP hybridfor Drug
Delivery Protein aggregation towards target cellsConversion :Light
energy Thermal energyCurcumin boundProtein with GNPSigma Xi Student
Research Showcase2
3. Layout of Project :Sigma Xi Student Research Showcase 3
4. Elastin-Like Polypeptide (E) - InverseTemperature
Transition(VPGXG)n Elastin-Like Polypeptide (E) Repeats 5 Amino
Acids X: Guest Residue; any Amino Acid but not Proline Possess
Inverse Temperature TransitionPossible mechanisms for the phase
transition of (VPGIG)40 proposed by Tirrell D.Chilkoti Group
Website. http://www.chilkotilab.pratt.duke.edu/researchELP.htm
(accessed January 6, 2012).Yamaoka, T.; Tamura, T.; Seto, Y.; Tada,
S.; Hunugi, S.; Tirrell, D. A. Biomacromolecules, 2003, 4,
1680-16854Sigma Xi Student Research Showcase
5. Coiled-Coil Domain of Cartilage Oligomeric Matrix Protein
(C) - Binding COMP 4 epidermal type 3 globulargrowth factor repeat
domain COMPcc or C CC(EGF) (T3) (TC) H2NCOOH2~6 ~30 73 VA, VD,
Curcumin, Ca2+, Cl-AnaMar Website.
http://www.compelisa.com/2/en/about-comp/what-is-comp.php (accessed
December 19, 2012).Ozbek, S.; Engel, J.; Stetefeld, J. The EMBO
Journal, 2002, 21, 5960-5968 Sigma Xi Student Research
Showcase5
6. Generate Smart Biomaterials Using E and C asBuilding
BlocksSigma Xi Student Research Showcase 6
7. DNA Transformation -PLASMID MAP PQE-30 LAC OPERON ANTIBIOTIC
RESISTANCEE 1C HIS-TAG3806 bpGLYCEROL STOCK XL1-BlueELECTROPORATION
AF-IQ Cells CE1 3816 bp Sigma Xi Student Research Showcase 7
8. Cell Expression -Streak Colonies Small scale Expression
Large scale ExpressionE1CCE1 glycerol stocksSuspend culture Colony
pick-upstreaked on TSAAMP platein 1L LB and suspension in Luria
Broth Incubation (370C) Induction with IPTG Overnight
IncubationOvernight incubation (370C)37oC Overnight
Incubation(370C)Sigma Xi Student Research Showcase 8
9. SDS PAGE Gel- Expression GelOsmotic Shock Gel E 1CCE1Sigma
Xi Student Research Showcase 9
10. Extraction and Purification - Cell Lysis with Sonication
30% impulse 2 mins 15 seconds Centrifugation to separate cell
lysates and ProteinsPurification using HPLC Ni Resin Column His
residue of protein binds to Niions. Elution collected with
differentpercentage of Buffer B(1%,5%,100%) Buffer A: 6 M urea, 50
mM Na2HPO4, 20 mM imidazole, pH 8.0 Buffer B: 6 M urea, 50 mM
Na2HPO4, 200 mM imidazole, pH 8.0 Sigma Xi Student Research
Showcase 10
11. Purification Result -E1CCE115 Kb 15 KbMW : 14.1 KbMW: 13.9
Kb Sigma Xi Student Research Showcase11
12. Quantification using MicroBCAPrinciple :Reduction of Cu to
Cu1+: peptides containing three or more amino acid residues form a
colored chelate 2+complex with cupric ions in an alkaline
environment containing sodium potassium tartrate.Bicinchoninic acid
(BCA) reaction : chelation of two molecules of BCA with one cuprous
ion to givepurple color. Protocol Standards of BSA were made of
concentration 40,20,10,5,2.5 ul On Microtiter plate - Std : 150 ul
+ 150 ul Reaction mixture Sample 15 ul + 135 ul of PB + 150 ul
Working reagent (A:B:C 50:48:2) 2 hrs of incubation at 37
C.http://www.qcbio.com/pierce/BCA%20Protein%20Assay%20Reagent.htmSigma
Xi Student Research Showcase 12
13. Result : BSA Standard Absorbance Plot Concentration
Calculation - CE1 E1CugmL223.3491705 141.420705 mgmL 0.223349171
0.14142071 MW 13941.72 13864.66molarity 1.60202E-05 1.02001E-05
Sigma Xi Student Research Showcase13
14. Integration of InorganicMaterials with Organic Protein
Polymers 14
15. Controllable Metal Nanostructures AuAg alloyAu
nanoparticles withnanosphere increasing Au concentration Au
nanorods ofAu increasing aspect nanorodsratio AgAg nanoprisms with
nanoprismsincreasing lateral sizeLiz-Marzn, L.M. Materials Today,
2004, 7, 26-31Sigma Xi Student Research Showcase 15
16. Potential of Gold Nanoparticles Based on Protein-Gold
Nanoparticles InteractionProtein adsorptionon GNPs surfaceProtein
misfolding Protein aggregationZhang, D.; Neumann, O.; Wang, H.l
Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.;
Wittung-Stafshede, P.; Halas, N.J. Nano Letters, 2009, 9, 666-671
Sigma Xi Student Research Showcase16
17. Process of Templation - incubate with HAuCl43H2O for 5 mins
40:1 molar ration between Au andprotein reduce with NaBH4 2.5:1
ration to Au4HAuCl4 + 3NaBH4 -> 4Au + 6H2 + 3NaCl + 3BCl3 + 4HCl
Gold binds to Histidine Uniform coatingSlocik, J. M.; Wright, D. W.
Biomacromolecules, 2003, 4, 1135-1141.Sigma Xi Student Research
Showcase 17
18. Overview on Circular Dichroism -a form of light
absorptionspectroscopy that measures thedifference in absorbance of
right-and left-circularly polarized light bya chiral moleculeSigma
Xi Student Research Showcase 18
19. Circular Dichroism for Proteins All amino acids except
glycine are chiral Phenomena exhibited due to e- transition n ->
* centerd around 215-230 nm ; involves non-bonding electrons of O
of the carbonyl -> * centerd around 185- 200 nm involves the
-electrons of the carbonyl The intensity and energy of these
transitions depends on and (i.e., secondary
structure)http://www.informatics.indiana.edu/predrag/classes/2008springi619/week4_m.pdf
- Andrs Szilgyi Sigma Xi Student Research Showcase 19
20. Study-1 : Impact on Secondary Structure of P and
PGNP1500025 CE1-GNP CE1GNP10000mrw (degCM2dmol-1) CE1wt CE1 GNPs
decrease the CD reading E1C-GNP E1CGNP 5000E1Cwt E1 C 0 GNPs
slightly increase the unordered structure for both E1C and-5000 CE1
-10000 180200 220 240 260Wavelength (nm)Sigma Xi Student Research
Showcase20
22. Study 2 : Impact of GNPs on Tt 1Uniformed Turbidity at 800
nm0.9E1C0.8 27 0.70.6 E1C-GNP E1C0.530 E1C-GNP CE10.4CE1-GNPCE1
CE1-GNP0.3 59 67 0.20.1 0020 40 60 80 100Temperature () Comparison
between Tts of E1C and CE1 are consistent with library dataTt of
both E1C and CE1 are enhanced after templation Enhanced Tt of CE1
after templation is greater than that of E1CSigma Xi Student
Research Showcase 22
23. Study 3 : Effect on Binding Ability Why Curcumin ?
Hydrophobic natureCOMP domain forms hydrophobic pore which aids
bindingof curcuminAnti-oxidant and anti-inflammatory
propertiesPotential for cancer treatmentAggarwal, B. B.; Shishodia,
S. (2004). "Suppression of the Nuclear Factor-B Activation Pathway
by Spice-Derived Phytochemicals: Reasoning forSeasoning".Annals of
the New York Academy of Sciences1030: 434441 Sigma Xi Student
Research Showcase 23
24. Result CCM to protein bindingProtein Samplemolar ratio
CE11.60.9 : 1 E1C1.80.8 : 1CE1GNP 2.10.7 : 1E1CGNP 2.50.6 : 1 GNPs
maintain the binding and releasing ability of E1C and CE1 to
CurcuminSigma Xi Student Research Showcase 24
25. Conclusions.. GNPs have impact on proteins conformation.
GNPs also affect on the thermoresponsive properties of the
proteins. The effect is greater on CE1 than E1C. GNPs do not
eliminate binding ability of the proteins.GNPs have influence on
proteins conformation andthermoresponsivity while still maintain
the binding ability of theproteins. Through templation it is
possible to control thethermoresponsivity of the proteins.Sigma Xi
Student Research Showcase 25
26. Thank you! and many morelab members!Sigma Xi Student
Research Showcase 26