protein-gepi fusion constructs protein-gepi adsorption on solid surfaces selection of specific gepis...
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Protein-GEPI Fusion Constructs
Protein-GEPI Adsorption on Solid Surfaces
Selection of Specific GEPIs
Specific GEPIs-directed Immobilization
pMAL
Clo
ne
Express
Recombinant GEPI-MBP
mal E MCS lacZ
Linker AuBP1
QBP2 Linker
MBP-QBP
MBP-AuBP
-WAGKRLVLREE
-LPDWWPPPQLYH
LinkerAuBP2
RFP-AuBP
-WALRRSIRRQSYRFP
Cleave
ITU UW IRES Joint Projects
FliTrx Peptide Display
Flagellin FlagellinThioredoxin
Flagellar filament
pFliTrx
Peptide domain
Randomized Constrained
PeptideGEPIs Syntheses
Biopanning experiment
Selection of Specific GEPIs
GEPIs Binding Affinity
WT Protein:
GEPI Fusion Protein:
Au Glass Au Glass
Glass Au GlassAu
Substrate
Binding Adsorption: SPR, LSPR, FM, AFM, QCM
Substrate
Ad) Protein-GEPI Fusion Constructs (DsRFP)
pMAL
Clo
ne
Express
Recombinant GEPI-MBP
mal E MCS lacZ
LinkerAuBP2
RFP-AuBP
-WALRRSIRRQSYRFP
Cleave
Outlines:
-Construction of the gene coding DsRFP-AuBP2) by general molecular biology tools and clonning into pMAL expression vector
- Expression of MBP-RFP-GEPI fusion proteins and affinity chromatography purification of recombinant MBP-RFP-GEPI fusion proteins
- Cleavage of MBP tag by Xaa cleaving factor
- Confirmation of produced RFP-GEPI fusion proteins molecular weight on SDS page gel electrophoresis
- Calculation of produced RFP-GEPI fusion proteins using Bradford assay
- Studies on RFP-GEPI fusion proteins adsorptions on solid surfaces using uCP, ELISA, SPR
Results: Agarose gel electrophoresisA) Construction of the DsRFP-AuBP2 gene
M C 2P 1P
M: marker, C: control, 2.P: second PCR product, 1.P: first PCR product. PCR made with two different
primers which are parts of AuBP sequence.
B) Clonning the DsRFP-AuBP2 gene in pGEMT easy clonning vector
M 1Cp 1Up 2Cp 2Up 3Cp 3Up 4Cp 4Up
M: Marker,Up: Uncut plasmid, Cp: cut plasmid.~720bp product observed. DNA sequencing confirmed that construct is in the vector.
Ad) Protein-GEPI Fusion Constructs (MBP)
pMAL
Clo
ne
Express
Recombinant GEPI-MBP
mal E MCS lacZ
Linker AuBP1
QBP2 Linker
MBP-QBP
MBP-AuBP
-WAGKRLVLREE
-LPDWWPPPQLYHM 1 2 3 4 5 6 7 8 M
M 1 2 3 4 5
Results: SDS PageA) Expression of MBP-QBP2
M – Marker1 – Uninduced cells2 – Induced cells3 – Flow through4 – 1st wash
5 – 3rd wash6 – Eluent (MBP-SGGG-QBP2)7- Eluent (MBP-PGPGPG-QBP2)8 – MBP WTM - Marker
B) Recombinant MBP-GEPI fusion proteins
MB
P-W
T
MB
P-(P
G)3-A
uB
P1
MB
P-S
(G)3-A
uB
P1
MB
P-(P
G)3-Q
BP
2
MB
P-S
(G)3-Q
BP
2
Ma
rke
r
Outlines:
- Expression of MBP-GEPI fusion proteins
- Affinity chromatography purification of recombinant MBP-GEPI fusion proteins
- Concentration of MBP-GEPI fusion proteins using ultrafiltration
- Confirmation of produced MBP-GEPI fusion proteins molecular weight on SDS page gel electrophoresis
- Calculation of produced MBP-GEPI fusion proteins using Bradford assay
- Studies on MBP-GEPI fusion proteins adsorptions on solid surfaces using uCP, ELISA, SPR
Ad) Protein-GEPI Adsorption on Solid Surfaces
WT Protein:
GEPI Fusion Protein:
Au Glass Au Glass
Glass Au GlassAu
Substrate
Binding Adsorption: SPR, LSPR, FM, AFM, QCM
Substrate
Results: FM experiments at ITUA) uCP - MBP-QBP2 on silica glass
: QBP-MBP (Printing)
PDMS
Y
: anti-MBP-Alexa
: BSA (Blocking)
Glass
Glass
Y
Glass
Y YY YY Y
Glass
Protocol: Micro Contact Printing
NC – no protein MBP-WT
MBP-(PG)3-QBP2 MBP-S(G)3-QBP2
B) uCP - MBP-AuBP1 on gold- in progress
Results: SPR experiments at ITU- in progress
Results: FM experiments at UWA) uCP - MBP-QBP2 on silica glassB) uCP – MBP-AuBP1 on gold- Completed (GEMSEC poster)
Summary:
MBP-GEPI fusion proteins:-MBP-GEPI fusion proteins were successfully expressed and purified
-uCP and FM techniques were optimized at ITU
-uCP and FM studies of MBP-QBPs adsorption to silica surface were performed (ITU and UW)
-uCP and FM studies of MBP-AuBPs adsorption to gold surface are in progress (ITU and UW)
-ELISA detection of MBP-AuBPs adsorption to gold surface was adapted and optimized at ITU (in progress)
DsRFP-GEPI fusion proteins:-DsRFP-AuBP2 was successfully constructed and prepared for clonning into the pMAL vector (ITU)
-Clonning DsRFP-AuBP2 into pMAL vector is in progress (ITU)
-Expression/Purification of DsRFP-AuBP2 is ongoing (ITU and UW)
-Protein adsorption and nanophotonic studies of DsRFP-AuBP2 will be performed at both facilities (ITU and UW)
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