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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
Proteins
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
Introduction
unstable or looses its fluorescence under Membrane Adsorber
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 Protein Analysis
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
References
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 Conclusion
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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