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)