Purification of His6-tagged Proteins
Metal Chelate Chromatography with Sartobind® Membrane Adsorbers
Materials and Methods
Production of Bgl-His
E.coli BL21 (DE3) pET-bgl-His6-sec was
cultivated in 300 ml medium (10 g/l peptone,
Green fluorescent protein (GFP) and β-glu-
10 g/l yeast extract, 20 g/l glycerol, 10 g/l
canase were used, which were fused with His6
NaCl, 100 mg/l ampicillin) in a 1 l-baffled
flask. One milliliter of the culture was inocu-lated and incubated overnight at 37°C on the
GFP is commonly used as reporter protein.
shaker. The induction with IPTG was carried
During the autocatalytic process, GFP forms
out after 2 hours. The cells were harvested
a structure that emits green light at 508 nm
by centrifugation at 11,000 x g for 30 min.
if stimulated by light with wavelength of
A part of the supernatant was passed through
a 0.2 µm filter and the filtrate was used for
maximums). Therefore it is relatively easy
to follow GFP during processing by means of fluorescence light. However, it becomes
Preparation of Sartobind IDA
unstable or looses its fluorescence under
certain conditions. GFP is an intracellular
Each Sartobind IDA unit containing 9.3 cm2
protein produced in E.coli that has to be
adsorption area (3 + 3.1 cm2 membrane disks
put into a filter holder) was flushed with
10 ml of deionized water, which was slowly
histidine (His6)* residues can be purified by
metal chelate chromatography. Metal chelate
chromatography is based on the complexing
water was allowed to run by gravity. The
of metal ions as Ni2+, Cu2+ or Co2+ immobi-
et al., 1990]. This fusion was cloned into a
following solutions were passed through in
lized by chelate formation through imidazole
pET20b+ vector, while the signal sequence
ring of the amino acid histidine. The bound
of β-glucanase was preserved. Transcription
His6-tagged* protein can be eluted from the
in E. coli was carried out by the T7 promoter
membrane selectively with imidazole, which
which was induced by IPTG. A secretion cas-
competitively binds to the metal complex.
sette is located on the plasmid. It consists of
2. 0.01 M Imidazol in 0.025 M KPO4, pH 8
Sartobind IDA enables to immobilize suitable
the gene for the bacterial release protein (kil)
subsequent purification. For evaluation the
phase promoter fic [Miksch et al., 1997]. The
parameters as capacity, effectiveness and
moderate expression of kil allows the secre-
tion of the glucanase from the periplasm into
Purification of Proteins
the medium. The isolation and purification of
the filtered cell-free supernatant of Bgl-Hg
carried out after removal of E. coli cells.
(80 ml) were applied to the Sartobind IDAunits. The flow-through was collected.
Production of His6-tagged GFP (His6-GFP)
The units were washed twice with 10 ml of
E.coli BL21 pHis6-GFP was cultivated under
the same condition as the glucanase and the
cells were separated by centrifugation. The
fractions were collected together. The pro-
pellet (23.3 g) was suspended in approx. 10 ml
teins were eluted twice with 3 ml of elution
of lysis buffer (50 mM NaH2PO4, 300 ml NaCl,
10 mM imidazole, pH 8.0, lysozyme 1 mg/l),
250 mM imidazole, pH 8.0). Each eluate was
collected separately. All obtained fractions
buffer was added. The cells were disrupted in
ice bath using pulsed ultrasound. The lysatewas centrifuged. After removal of 5 ml
Polyacrylamide gel electrophoresis (PAGE)
through a 0.2 µm filter. The filtrate was used
and estimation of fluorescence of GFP and
enzyme activity of Bgl were carried out.
Results and Discussion
Borris, R., Buettner, K. and Maentsaelae, P.
Lysate (lane 2, 3) and flow through (lane 4)
show plenty of bands of intracellular proteins.
Molecular & general genetics 222 (2-3),
Only the bands of the proteins in high con-
centrations are visible in wash fraction (lane5). Eluate 1 (lane 6) shows a clear band below
Miksch, G., Fiedler, E., Dobrowolski, P.,
Fries, K. (1997): The kil gene of the ColE1
27.7 kD. The two other bands are intracellular
plamid of Escherichia coli controlled by a
proteins that were also bound. It is common
occurrence at lysate purification. Eluate 2
the secretion of a heterologous periplasmic
(lane 7) shows no band what indicates all
proteins have been eluted in the first step.
Arch. Microbiol. 167 (2+3), 143-150.
This Application Note is courtesy of Friehs K,
University Bielefeld: Isolierung rekombinanter
Proteine mit Membranmodulen. Bericht.
4 Flow-through5 Wash fraction6 Eluate 17 Eluate 28 Size Standard
The samples of supernatant and flow throughwere concentrated. These lanes show theclear broad bands of the glucanase. There isstill the very broad band in the flow-throughwhat suggests the concentration of the glu-canase was too high to isolate entire protein.
The wash fraction (lane 5) show no visibleband, that means, the target protein bindingwas stable. Each eluate (lane 6, 7) shows oneband of the target protein. This indicates that the Membrane Adsorber unit was reallycompletely saturated with the target protein.
The transparent background demonstrates
additionally that there were no proteins in
The protein concentration in eluate 1 and 2
were 232 and 127 µg/ml, respectively. Since
3 ml of each were collected, the total amount
is 1077 µg. With assumption that a third
eluate would still contain some protein, the
represents a binding capacity of 118 µg/cm2
or 4.3 mg/ml (1 ml = 36.4 cm2 membrane).
Sartobind® is a trademark of Sartorius AG
The Sartobind IDA membrane charged withCu2+ are suitable for purification of both
Technical data are subject to change without notice.
intracellular and extracellular proteins.
Printed in Germany on paper that has beenbleached without any use of chlorine.
Publication No.: SL-4037-e04101Order No.: 85030-521-33
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