Transcript Expression and purification of membrane proteins: Initial

Expression and purification of
membrane proteins: Initial screening
of Thermotoga maritima α-helical
membrane proteins for NMR
structural studies
The membrane proteome of T. maritima
Electron micrograph of T. maritima.
The arrow points to the outer
membrane, the “toga”, for which the
organism is named.
Percentage of Membrane Proteins in T. maritima
Membrane proteins
24%
24%
76%
76%
Cytosolic proteins
Functional Annotations of the Membrane Proteome
ABC transporters
20%
20%
55%
55%
Proteins with
unknown function
25%
25%
Other functionally
annotated proteins
Overall Approach to Preparing Membrane Protein
Samples for NMR Studies
Target selection
Assay expression
in E.coli
Determine localization
Insoluble fraction
Refold
Optimize expression
to the membrane
Membrane fraction
Extract
Co2+ or Ni2+-affinity
purification
Detergent stability
screen
NMR
spectroscopy
Target Selection
NMR sample requirements
600 mL of 1 mM protein
uniform 15N and 13C-labeling
monodisperse
MW < 30 kD (for standard NMR experiments, development
of TROSY has extended the MW limit for NMR
studies)
Size distribution of the membrane proteome
80
70
60
Selected fifty targets less
than 16 KD (~130 aa) that
contained one to four
predicted transmembrane
segments.
50
# of 40
proteins
30
20
Protein length (amino acids)
1551
1501
1400
1451
1401
1351
1301
1200
1251
1201
1151
1101
1000
1051
951
1001
901
800
851
801
751
701
600
651
601
551
501
400
451
401
351
301
200
251
201
151
0
101
0
0
51
10
1600
Expression Assay
Pa
PT
ra
7
Expression Vector
6-aa 6-his
CAC GTG
Nco I
Pml I
TM gene
TTA ATT AA
96 x 65 mL Fermentor
RBS Sma I Pme I
Pac I
kD
21.5
14.4
6.0
Eleven out of fifty targets overexpressed using arabinose induction.
Localization of target membrane proteins
TM1554
IB
S
M
TM1634
IB
S
M
In order to verify that the target proteins
are membrane proteins, the E. coli
membranes
were
isolated
using
ultracentrifugation and extracted with ndecyl--D-maltoside .
Of the eleven expressing targets, two
expressed exclusively in the insoluble
fraction (IB) and nine expressed to both
the insoluble fraction and the n-decyl--Dmaltoside solubilized membrane (M)
fraction. None of the proteins were found
in the soluble (S) fraction.
Soluble, monodisperse, and folded
Is that too much to ask?
Which detergent will do all this? For now, there is no paradigm. There isn’t one
detergent that works for all membrane proteins; therefore, for each protein,
many detergents need to be screened.
Detergent Screen
NG
OG
P
S P
S
DG
P
OM
S P
DM
S P
S
TM0361
DoDM
DHPC CHAPS LDAO DPC
P
P S
S
P S P S P S
Aromatic side
Backbone chain protons
amide protons
W indole side
chain protons
1H
(ppm)
Soluble ≠ folded!
9.5
9.0
8.5
8.0
1H
7.5
(ppm)
7.0
6.5
Optimization of solution conditions for structure
determination: 2D spectroscopy of 15N-labeled protein
TM0361
105
TM1634
110
110
115
115
15N
120
(ppm)
15N
120
(ppm)
125
125
130
130
11.0
10.0
9.0
8.0
(ppm)
1H
7.0
9.0
8.5
8.0 7.5
1H (ppm)
7.0
Using the 96 x 65 mL fermentor, these samples were prepared with less than 1L of commercial media.
Summary
1. Approximately 20% of the membrane protein targets express in
HK100 E. coli cells with arabinose induction.
2. Detergent screening has been successful in preparing solubilized
membrane protein samples, but will be revised as we gain
experience.
3. Similar to soluble proteins, 1H 1D NMR spectroscopy is suitable for
evaluating the overall fold of the protein.
4. TM1634 has been optimized and NMR structure determination is in
progress.
Acknowledgements
Kurt Wüthrich
Scott Lesley
Heath Klock
Bernhard Geierstanger
Joanna Hale
This work is funded by the NIH protein structure initiative grant P50 GM62411. LC is
funded by NIH grant 1F32GM068286. KW is funded by the endowment of Cecil H. and
Ida M. Green (TSRI).