Team:Bielefeld-Germany/Protocols/Production
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===His-tag affinity chromatography=== | ===His-tag affinity chromatography=== | ||
+ | * For buffers see [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography here] | ||
====Syringe method==== | ====Syringe method==== | ||
- | [[File:Bielefeld2012_Chromatography_Columns.JPG|300px|thumb|right|Used Chromatographycolumns: | + | [[File:Bielefeld2012_Chromatography_Columns.JPG|300px|thumb|right|Used Chromatographycolumns: |
- | (left) 1 mL HisTrap FF crude by GE Healthcare, | + | (left) 1 mL HisTrap FF crude by GE Healthcare, |
- | (middle) 15 mL HisTrap FF crude by GE Healthcare, | + | (middle) 15 mL HisTrap FF crude by GE Healthcare, |
(right) TALON His-Tag Purification Resin by Clonetech]] | (right) TALON His-Tag Purification Resin by Clonetech]] | ||
* Column: 1 mL HisTrap FF crude by GE Healthcare | * Column: 1 mL HisTrap FF crude by GE Healthcare | ||
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====ÄKTA method==== | ====ÄKTA method==== | ||
- | |||
* Column: 15 mL HisTrap FF crude by GE Healthcare or | * Column: 15 mL HisTrap FF crude by GE Healthcare or | ||
- | * If Column is not loaded with Ni-ions: | + | * If Column is not loaded with Ni-ions /Cobalt-ions: |
**Wash column with 50 - 80 mL of deionized water | **Wash column with 50 - 80 mL of deionized water | ||
- | **Load column with 1,4% NiSO4-Solution | + | **Load column with 1,4% NiSO4-Solution or CoCl<html><sub>2<sub/><html/> |
* Wash column with 50 - 80 mL of deionized water | * Wash column with 50 - 80 mL of deionized water | ||
* Equilibrate column with 50 - 80 mL of binding buffer | * Equilibrate column with 50 - 80 mL of binding buffer |
Revision as of 18:57, 20 September 2012
Contents |
Production
Here all our methods according to cultivation and purification are listed.
Cultivation
Expression of Laccase
- Chassis: Promega's E.Coli KRX
- Medium: Autoinduction-Medium supplemented with Chloramphenicol (final concentration 60 μg mL-1)
Cultivation with E. coli KRX in shaking flask(with baffles):
- 200 mL culture in 1000 mL shaking flask with baffles (Schott) with silicon plugs
- Cultivation temperature: 37 °C
Autoinduction-medium with 20-60 mg L-1 chloramphenicol and if nessassary with 100-300 mg L-1 ampicillin
- Shaking at 140 rpm
- for characterizations: automatic sampling every 30 min
Bioreactor cultivations with E. coli KRX
To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor. It is possible to cultivate several liters and to control temperature, pH and pO_2.
- Bioreactor: Braun Biostat B Bioreactor (3L), Infors Labfors Bioreactor (3L), Bioengineering NLF22 Bioreactor (7 L),
- Autoinduction-medium with 60 mg L-1 chloramphenicol
- Culture volume: 3,0-6,0 L
- Starting OD600: 0.1 - 0.2
- Airflow: 5 NL/min
- pO2-Control: 30 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
- pO2=100% calibration with 300rpm
- pH: 7.0 (controlled with 2M phosphoric acid and 2 M NaOH)
- Antifoam: BASF pluronic PE-8100
- Harvest after 12-13 h
Purification
His-tag affinity chromatography
- For buffers see here
Syringe method
- Column: 1 mL HisTrap FF crude by GE Healthcare
- Equilibrate with binding buffer(10mL)
- Load sample onto column(max. 6 mL)
- Wash with 10 mL binding buffer
- Elute with 5 mL of elution buffer
- Collect the eluate in 1 mL fractions, the purified protein is most likely in the first or second fraction
- Re-equilibrate the column with binding buffer
ÄKTA method
- Column: 15 mL HisTrap FF crude by GE Healthcare or
- If Column is not loaded with Ni-ions /Cobalt-ions:
- Wash column with 50 - 80 mL of deionized water
- Load column with 1,4% NiSO4-Solution or CoCl2 * Wash column with 50 - 80 mL of deionized water * Equilibrate column with 50 - 80 mL of binding buffer * Load column with supernatant of the lysed cells (Collect the Flow through for SDS-PAGE analysis) * Wash Column with 50 - 80 mL of binding buffer (Collect the Flow through for SDS-PAGE analysis) * Elute Protein with an increasing elutionbuffer ratio (gradient 0%-100%, length 200mL) * Collect the eluate in 10 mL fractions * Elute remaining proteins with 100% Elutionbuffer (40mL)