Download Standardized solubilization and purification of different

Standardized solubilization and purification of different
membrane proteins from E. coli, rat, and human
Jan Kubicek1, Christa Broermann1, Antje Schütz1, Jutta Drees1, Nicole Brinker1, Barbara Maertens1, Helena Block1,
Anne Spriestersbach1, Jörg Labahn2, and Frank Schäfer1
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
2
Institute of Structural Biology and Biophysics (ISB-2), Research Center Jülich, 52425 Jülich, Germany
1
Membrane proteins — interesting, but challenging
Purification of NhaA from E. coli
Approximately 30% of genes encode for membrane proteins, which in turn
represent most of the investigated drug targets. Membrane proteins constitute
~50% of possible targets for novel drugs and ~70% of currently marketed
Screening for the optimal detergent for solubilization
Solubilization procedure
and purification of a homologue-expressed protein NhaA
form E. coli. NhaA was best solubilized using DDM.
Supernatant from
lysed cells from E. coli
or Insect cells
drugs act on G protein-coupled receptors and ion channels alone.
A
kDa
However, handling membrane proteins is challenging, with very few membrane
protein structures solved to-date. Membrane proteins have large hydrophobic
areas and tend to aggregate; hence, detergents are needed to solubilize them.
However, the variability of membrane proteins makes it difficult to predict the
Pellet in detergent
shake 1h
M
OG
DM
LDAO DDM
Cy6
NG
FOS
80 —
60 —
NhaA
40 —
NhaA
M
TP
SF
FT
W
E
kDa
72 —
30 —
right detergent for solubilization and purification. Factors such as size, number
of transmembrane domains, and ionic strength are crucial for effective
solubilization and purification of a specific protein. Therefore, different detergents
are suitable for solubiliziation of different membrane proteins.
Subsequent purification was performed using Ni-NTA
Superflow with the addition of DDM. Fractions were
separated on an SDS-PAGE gel and proteins visualized
by Coomassie® staining.
55 —
20 —
NhaA
43 —
15 —
Supernatant: soluble
protein in detergent
34 —
B
kDa
To facilitate the screening for the optimal detergent for protein solubilization
and purification, we have developed the Ni-NTA Membrane Protein Kit. This
kit with a set of 7 well selected detergents enables standardized handling of
membrane proteins. The complete procedure of solubilization and purification
M
OG
DM
LDAO DDM
Cy6
NG
FOS
NhaA
26 —
80 —
60 —
NhaA
40 —
NhaA
30 —
can be reproducibly scaled up (data not shown) using Ni-NTA Superflow and
the same detergents in bulk.
17 —
20 —
Soluble protein in detergent
(Apply the same procedure
for all 7 detergents in parallel)
15 —
11 —
The indicated detergents were used to solubilized proteins from A the total protein
fraction and B the detergent-soluble membrane fraction from cells overexpressing the
35 kDa E. coli membrane protein NhaA (arrowed). Fractions were separated on an
SDS-PAGE and proteins visualized by immunodetection of the His-tag after western
blotting. M: His-tagged marker proteins. Part B shows that DM, LDAO, DDM, Cy6 and
Fos-16 are suitable for solubilization of this protein. The band at 70 kDa is a protein dimer.
TP: total protein; SF: soluble fraction; FT: flow-through fraction; W: wash fraction;
E: eluate; M: markers.
Purifying mammalian OGCP from insect cell (Sf9 cells) culture
Solubilization of the human membrane protein Presenilin
Rat mitochondrial-2-oxoglutarate/malate Carrier Protein (OGCP, 34 kDa) expressed in insect cell was purified using
Ni-NTA Superflow and buffers containing DDM. The Ni-NTA Membrane Protein kit was used to screen for the most
suitable detergent. Fractions were separated on an SDS-PAGE gel and proteins visualized by immunodetection of the
The membrane protein, Presenilin, is involved in important cellular processes in humans and defects are implicated
in the development of Alzheimer’s disease. The detergents DM, LDAO, DDM Cy6 and FOS are suitable for
solubilization of Presenilin. The Ni-NTA Membrane Protein Kit was used to screen for the most suitable detergent for
solubilization of the human membrane protein Presenilin. The protein was expressed in E. coli C41(DE3) solo cell
culture using an expression-optimized QIAgenes® E. coli construct.
His-tag after western blotting. Purification with Ni-NTA Superflow was performed with the detergent DDM.
120
80
60
50
40
N
G
M
kD
170
130
95
72
55
43
FO
S
LD
AO
DD
M
Cy
6
N
G
B
Soluble protein fraction
FO
S
O
G
LD
AO
DD
M
Cy
6
M
DM
kDa
O
G
Total protein fraction
DM
A
—
—
—
—
—
TF
SF
FT
W
E1 E2
E3 E4
E5
The 52 kDa protein Presenilin was expressed in TB-medium for 16 hours at 18°C and induced using 1 mM isopropyl-
E6
beta-D-thiogalactopyranoside (IPTG). The protein expressed well and could be best solubilized using the detergent
Fos-choline 16. Scale-up of expression and protein purification is currently ongoing.
—
—
—
—
—
—
M
34 —
OGCP
SDS OG DM LDAO DDM Cy6 NG FOS
kDa
26 —
30 —
Presenilin
50 —
OGCP
20 —
17 —
40 —
15 —
30 —
C
kD
The indicated detergents were used to solubilize proteins in the total protein fraction.
B Purification using Ni-NTA Superflow was performed with the addition of detergent DDM,
identified in the screening to be required for successful solubilization. Fractions were separated
by SDS-Page and visualized by Coomassie® staining. SDS-PAGE gel. TF: total fraction;
SF: soluble fraction; FT: flow-through fraction; W: wash fraction; E: eluate; M: His-tagged marker
proteins. C Fractions were separated on an SDS-PAGE and protein OGCP 34 kDa visualized
by immunodetection of the His-tag after western blotting. TF: total fraction; SF: soluble fraction;
FT: flow-through fraction; W: wash fraction; E: eluate; M: His-tagged marker proteins.
M
TF
SF
FT
W
E1 E2
E3 E4
E5
E6
E7
A
80
60
50
40
Fractions were separated on an SDS-PAGE gel and proteins visualized by
immunodetection of the His-tag after western blotting. The indicated detergents were
used to solubilize proteins.
—
—
—
—
30 —
OGCP
20 —
15 —
Cell-free expression and solubilization of Cav-1 from human
Conclusions
The His-tagged human Caveolin-1, a 20 kDa membrane-anchored protein, was expressed cell-free using an expressionoptimized QIAgenes Insect/Mammalia construct in EasyXpress Insect Kit II lysates. Detergent screen and purification
We analyzed the literature with regard to detergents used for solubilization and purification of recombinant His-tagged
membrane proteins. Approximately 50 detergents were experimentally screened for recovery of more than
10 prokaryotic and eukaryotic targets of different membrane proteins types (α-helical, β-barrel etc.) and from the
were performed using the Ni-NTA Membrane Protein Kit.
Total Fractions
A
M
NhaA
OG
DM
LDAO
DDM
membrane fraction of various expression systems. We have identified a panel of 7 high-quality detergents, at least
one of which allowed membrane protein purification in each test case. Based on the methodology a kit has been
Soluble Fractions
Cy6
NG
FOS
OG
DM
LDAO
DDM
Cy6
NG
FOS
kDa
developed which comprises the following features:
■ Membrane proteins from different origin expressed in E. coli, insect cells, or cell-free can be successfully
solubilized and purified.
50 —
40 —
■ A standardized solution for membrane protein solubilization and purification is available and eliminates
30 —
tedious evaluation of a large set of detergents.
20 —
Cav-1
■ The complete process can be reproducibly scaled-up.
15 —
Purification using Ni-NTA Superflow was performed with
the addition of detergent DM, identified in the screening
to be required for successful solubilization.
Fractions were separated by SDS-PAGE and proteins visualized by immunodetection
of the His-tag after western blotting. The detergents indicated were used to solubilize
proteins in the total protein fraction. NhaA: 35 KDa positive control. B Fractions were
separated by SDS-PAGE and proteins visualized by immunodetection of the His-tag after
western blotting. E: eluate; M: His-tagged marker proteins. NhaA: 35 KDa positive control;
TF: total fraction; SF: soluble fraction; FT: flow-through fraction; W: wash fraction.
xxxxxxx
09/2009
A
■ All proteins were expressed using QIAgenes (optimized expression constructs) as templates.
B
NhaA
M
TF
SF
FT
W
E1
E2
E3
■ Detergents in bulk amounts – with the same quality as in the Ni-NTA Membrane Protein Kit are also available.
E4
kDa
50 —
40 —
30 —
20 —
Cav-1
Hoffmann-La Roche owns patents and patent applications pertaining to the application of Ni-NTA resin (Patent series: RAN 4100/63: USP 4.877.830,
USP 5.047.513, EP 253 303 B1), and to 6xHis-coding vectors and His-labeled proteins (Patent series: USP 5.284.933, USP 5.130.663, EP 282 042 B1). All
purification of recombinant proteins by Ni-NTA chromatography for commercial purposes, and the commercial use of proteins so purified, require a license from
Hoffmann-La Roche.
15 —
10 —
Trademarks: QIAGEN®, QIAgenes®, EasyXpress® (QIAGEN Group); Coomassie® (ICI [Imperial Chemical Industries] Organics Inc.).
© 2009 QIAGEN, all rights reserved.
Sample & Assay Technologies