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From 2012.igem.org

Contents 1 Production 1.1 Cultivation 1.1.1 Expression of Laccase 1.1.1.1 Cultivation with E. coli KRX in shaking flask(with baffles): 1.1.1.2 Bioreactor cultivations with E. coli KRX 1.2 Purification 1.2.1 His-tag affinity chromatography 1.2.1.1 Syringe method 1.2.1.2 ÄKTA method 1.2.1.2.1 Column preparation 1.2.1.2.2 Chromatography protocol for the Äkta-system

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

• Columns:

1. 15 mL HisTrap FF crude by GE Healthcare
2. 50 mL TALON-Histag-Purification Resin by Clonetech

Column preparation

• If Column is not loaded with Ni-ions /Cobalt-ions:
  • Wash column with 5 - 8 Columnvolumes (CV) of deionized water
  • Load column with metal-ions(4 CV)
    • For HisTrap FF crude: 1,4% NiSO₄-Solution
    • For TALON-Histag-Purification Resin: CoCl₂-Solution

Chromatography protocol for the Äkta-system

• Wash column with 10 CV of deionized water
• Equilibrate column with 10 CV of binding buffer
• Load column with supernatant of the lysed cells (Collect the Flow through for SDS-PAGE analysis)
• Wash Column with 10 CV 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 (4 CV)