Purification and Chromatographic Characterisation of an Integral Membrane
Protein for Three-dimensional Crystallisation
Lars Haneskog, Jeffry Abramson* and So Iwata*.
Research and Development, Amersham Biosciences, S-751 84 Uppsala, Sweden
*Department of Biochemistry, Biomedical Center, Uppsala University, Box 576, S-751 23 Uppsala, Sweden
Abstract
• A histidine-tagged integral membrane protein was purified by
immobilised metal ion affinity chromatography.
• The charge and size homogeneity of the obtained detergentprotein complexes was assessed by chromatography.
• Crystallisation trials were made.
Introduction
Membrane protein crystallography has until this date only resulted
in a handful of high-resolution structures. Growing three-dimensional
crystals of membrane proteins requires a pure and homogenous
preparation of the protein in detergent solution. We have used a new
ÄKTA®design chromatography system for purification of the histidinetagged cytochrome bo3 ubiquinol oxidase from Escherichia coli
over-expressed in E. coli in order to obtain crystallisable material.
Cytochrome bo3 was purified by immobilised metal ion affinity
chromatography and the purified protein was characterised for purity
and for homogeneity in charge and size of the detergent-protein
micelles. Crystallisation trials were made.
Materials and methods
Results and discussion
Purification
Charge characterisation
Size characterisation
Chromatography on HiTrap Chelating yielded two distinct peaks (Figure 1).
The material in both peaks had the characteristic colour of cytochrome bo3.
SDS-PAGE showed that both fractions contained the four subunits expected for
cytochrome bo3 (Figure 2). Fraction 1 gave additional bands indicating contaminations. Fraction 2 showed only traces of contaminants. Scaling-up five times
gave the same result and yielded about 20 mg in each fraction.
Anion exchange chromatography on Mono Q HR 5/5 in the
presence of octyl-b-D-glucoside showed that Fraction 1 was
homogenous by charge (Figure 3a) as revealed by a single peak,
although several small peaks were observed that may be caused
by the trace contaminants. Fraction 2 gave a single sharp peak
(Figure 3b) indicating high homogeneity by charge.
Gel filtration on Superdex HR 10/30 in the presence of octyl-b-D-glucoside showed that
Fraction 2 eluted in a narrower zone than Fraction 1 (Figure 4). The elution position
corresponded to a molecular weight of 160 000±5 000 for the detergent-protein complexes. This value is probably underestimated due to the dynamic structure and flexibility
of the detergent moiety of the complexes, but indicates that cytochrome bo3 is monomeric
(polypeptide molecular mass 130 000). The origin of the small unresolved peaks forming
shoulders on the main peaks is unknown, but may correspond to optical effects caused
by protein-free detergent micelles.
Column:
Eluent A:
Eluent B:
Mono Q HR 5/5
20 mM tris-HCl, pH 7.5, with 1 % octyl-b-D-glucoside
20 mM tris-HCl, pH 7.5, with 1 % octyl-b-D-glucoside,
1 M NaCl
Gradient:
0-50%, 20 column volumes
Flow rate:
1 ml/min
Temperature: 5°C
System:
ÄKTA FPLC with UNICORN control
Sample loop: Superloop 10 ml
a) Sample:
2.5 ml fraction 1-material diluted 10 times with eluent A
b) Sample:
3 ml fraction 1-material diluted 10 times with eluent A.
a)
HiTrap Chelating 1 ml, Ni2+-loaded
8.5 ml detergent extracts of Escherichia coli membranes
(see Material and methods)
Eluent A:
20 mM tris-HCl, pH 7.5, with 5 mM imidazol,
0.03% dodecyl-b-D-maltoside and 300 mM NaCl
Eluent B:
20 mM tris-HCl, pH 7.5, with 500 mM imidazol,
0.03% dodecyl-b-D-maltoside and 300 mM NaCl
Flow rate:
1 ml/min
Gradient:
0-60%, 20 column volumes
Temperature: 5°C
System:
ÄKTA FPLC with UNICORN control
Sample loop: Superloop 10 ml
Column:
Sample:
%B
mAU
Bacterial membranes were prepared essentially as described by Rumbley et al. (1)
and were solubilised by addition of 10 ml 1% dodecyl-b-D-maltoside, 300 mM
NaCl in 20 mM tris-HCl buffer, pH 7.5 per 1 g sedimented membranes followed
by stirring. Non-solubilised material was removed by centrifugation.
Purification was done on HiTrap® Chelating in the presence of the non-ionic
detergent dodecyl-b-D-maltoside, and characterisation was performed under
non-denaturing conditions by anion exchange chromatography on
Mono Q® HR 5/5 and gel filtration on Superdex® 200 HR 10/30 in the presence
of the non-ionic detergent octyl-b-D-glucoside. The exchange of detergent may
favour crystallisation, since small detergent micelles are generally found to be
more suitable than large. Chromatography was performed at 5°C.
50
100
40
80
30
60
20
40
100
1500
80
Fraction 2
1000
60
Fraction 1
40
b)
mAU
mS/cm
11.84
60
10
mAU
mS/cm
11.94
60
10
8
8
6
6
40
20
0
4
4
2
2
0
0
20
300 µl applied via 0.1 ml sample loop
20 mM tris-HCl, pH 7.5, with 1.5 % octyl-b-D-glucoside, 150 mM NaCl
0.25 ml/min
5°C
ÄKTA FPLC with UNICORN control
Fraction 1 from HiTrap Chelating 1 ml
Fraction 2 from HiTrap Chelating 1 ml
40
10
500
Superdex 200 HR 10/30
a)
%B
mAU
Column:
Sample
application:
Eluent:
Flow rate:
Temperature:
System:
a) Sample:
b) Sample:
10
20
ml
b)
0
0
0
10
20
30 ml
Figure 1. Purification on HiTrap Chelating columns
Preparation of E. coli cell membranes
100
50
0
5
10
15
20
ml
0
5
10
15
20
ml
Figure 4. Chromatographic characterisation on Superdex 200 HR 10/30.
40
1 2 3 4 5
60
30
Solubilisation of membranes with non-ionic detergent
Lane 1: Low Molecular weight Calibration kit (LMW); 14 400,
20 100, 30 000, 43 000, 67 000, 94 000
Lane 2: Detergent extract of E. coli membranes
Lane 3: Flow-through material
Lane 4: Fraction 1 from HiTrap Chelating 1 ml
Lane 5: Fraction 2 from HiTrap Chelating 1 ml
Analysis of:
Purity by SDS-PAGE
Charge homogeneity by ion exchange chromatography
Size homogeneity by gel filtration
0
0
80
Purification by HiTrap Chelating chromatography
20
20
%B
mAU
60
20
40
10
20
0
0
10
20
ml
0
Figure 3. Chromatographic characterisation on Mono Q HR 5/5.
Crystallisation
Figure 2. SDS electrophoresis on PhastSystem™ using PhastGel® 8-25%, silver staining.
Crystallisation trials revealed poor crystals on material passed
through Mono Q. Adjustments of the purification protocol is
ongoing.
Summary
Histidine-tagged cytochrome bo3 was efficiently purified in one step on
HiTrap Chelating, although two fractions were obtained. Both fractions were
essentially homogeneously monomeric by charge and size. Crystals were
obtained, but further improvements of the purification protocol or the crystallisation procedure have to be done in order to obtain crystals of suitable
quality for x-ray diffraction analysis.
Overwiew of the procedure
Acknowledgements
References
This work was supported by EU grant BIO4-CT97-2357 (DG12-SSMI) “New
approaches in the crystallisation and crystallography of membrane proteins”.
1. Rumbley, J. N., Furlong Nickels, E. and Gennis, R. B. (1997) One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli
and demonstration that associated quinone is not required for the structural integrity of the oxidase. Biochim. Biophys. Acta 1340, 131-142
2. Poster nr 39.
The following designations are trademarks own by Amersham Biosciences:
ÄKTA, FPLC, UNICORN, HiTrap, PhastSystem, PhastGel, Mono Q, Superdex.