Embed Size (px)
Citation preview
Characterisation of FeoB, the Iron (II) Transporter from Pseudomonas aeruginosa
SAEED SEYEDMOHAMMAD
DEPARTMENT OF PHARMACOLOGY
UNIVERSITY OF CAMBRIDGE
24/10/13
PROTEIN EXPRESSION, PURIFICATION AND CHARACTERISATION
BACTERIAL IRON ACQUISITION TRANSPORTERS ASPOTENTIAL DRUG TARGETS
Feo System
Fe (III) Transport
Siderophore-MediatedPyoverdine
Pyochelin
INTRO.
Feo Iron (II) Transport System
Iron acquisition facilitated via FeoB
GTPase activity
C-Terminal
N-Terminal
INTRO.
OBJECTIVES
Effect of FeoA on FeoB
GTPase Activity 8-Histidine
Tag
C41 (DE3)
Reconstituting in Liposomes
Purifying FeoA
Optimising FeoB overexpression
Purifying FeoB to Homogeneity
Aims
Expression & Stability
pET41-a (+)
Cloning of full-length feoB gene from
P. aeruginosa
Aim I
Aim II Cloning FeoA
I) Optimising FeoB Protein
Media
Temperature
Detergent
pH
Aggregation DLS CD GTPase
I - FeoB OVEREXPRESSION AND PURIFICATION
(A) Expression
(B) Purification
AIMS
I - FeoB OVEREXPRESSION
Coomassie Staining Western Blotting
FeoB
Mutant Equally expressed to Wild-type
RESULTS
I - OPTIMISING FeoB EXPRESSION
Expressed in E. coli C41 (DE3) Expression in Different Growth Media
Expression at Different Temperatures
FeoB expressed most in LB media 2x
YT
TBLBNon
-Exp
ress
ing
Con
trol
Non
-Exp
ress
ing
Con
trol
37°C
30°C
25°C
20°C
FeoB
FeoB expression increases with a reduction in temperature
RESULTS
I - FeoB PURIFICATION
GTPase Activity AssaySDS-PAGE
FeoB solubilised by DDM and purified by Ni-NTA Metal Affinity Chromatography
FeoB Exhibited GTPase Activity FeoB-D123N Defective in GTPase Activity
FeoB
RESULTS
I - CIRCULAR DICHROISM SPECTROSCOPY OF FeoB INDDM DETERGENT
Curve depicts predominantly α-helix
The secondary structure of FeoB is stabilised in DDM
RESULTS
DETERGENTS USED FOR PURIFYING FeoB
DDM
C12E8
Amphipol A8-35
NV10
I - FeoB AGGREGATION IN DIFFERENT DETERGENTS
10 % SDS-PAGE GTPase Activity Assay
FeoB least aggregated in C12E8 and Amphipol A8-35 Detergents
FeoB most aggregated in NV10
FeoB exhibited highest GTPase activity in DDM and lowest in NV10
RESULTS
I - EFFECT OF PH ON FeoB ACTIVITY & STABILITY
GTPase Activity Assay10 % SDS-PAGE
FeoB aggregation increases at lower pH
FeoB loses GTPase activity above pH 7.5
RESULTS
I - EFFECT OF DETERGENT ON FeoB AGGREGATION
FeoB aggregation highest in NV10 Polymer
FeoB fully stable in C12E8 and Amphipol A8-35 upon initial testing
RESULTS
10 % SDS-PAGE
2) Effect of FeoA on FeoB GTPase
Activity
FEOA INFLUENCE ON FeoB GTPASE ACTIVITY
B) In Reconstituted
A) In detergent Micelle
GTPase Activity
IMAC Purification
II - OPTIMISED EXPRESSION OF FeoA IN DIFFERENT BACTERIAL MEDIA AND TEMPERATURES
FeoA
FeoA predominantly expressed in the pellet as inclusion bodies
RESULTS
II - PURIFICATION OF FeoA FROM INCLUSION BODIES
FeoA purified to homogenity from both soluble and insoluble (IB) fractions
FeoA
RESULTS
II - GTPASE ACTIVITY OF FeoB
FeoB exhibited GTPase activity that is stimulated by FeoA
+ FeoB WT
RESULTS
Micelle
II - GTPASE ACTIVITY OF FeoB
FeoB exhibited GTPase activity when reconstituted in liposomes with FeoA
RESULTS
Liposome
Full-length feoB gene was successfully cloned from P. aeruginosa
FeoB expression levels optimised and purified to homogeneity
FeoB exhibited GTPase activity when the D123N FeoB mutant was defective in GTP binding
FeoA was successfully cloned, expressed and purified
Preliminary data showed the stimulatory effect of FeoA on FeoB GTPase activity in detergent micelles and when reconstituted in liposomes
CONCLUSIONS
Investigate the coupling reactions between FeoB GTPase activity and Fe2+ transport
Confirm the putative interaction between FeoA and FeoB
Reconstitute FeoB in lipid vesicles and directly measure FeoB-mediated iron transport in absence of other proteins, under controlled conditions
Assess whether FeoB membrane domain facilitates GTP-independent Fe2+ transport
FUTURE DIRECTIONS
ACKNOWLEDGEMENTS
• Dr. Rietie Venter• HV Lab
Collaborators
• Nelson Barrera – Pontificia Universidad Catolica de Chile
• Robinson Lab University of Oxford • Edwardson Lab University of Cambridge
