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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 Cell Harvesting 1.3 Mechanical lysis 1.3.1 Mechanical lysis of the (shaking flask) cultivation 1.3.2 Mechanical lysis of the (bio-reactor) cultivation 1.4 Purification 1.4.1 His-tag affinity chromatography 1.4.1.1 Syringe method 1.4.1.2 ÄKTA method 1.4.1.2.1 Column preparation 1.4.1.2.2 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

Cell Harvesting

• Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
• if the purification should start the next day store the cell pellet at 4°C !(the laccase must not be frozen!)
• Resuspend the pellet in 5 mL binding buffer (His-tag affinity chromatography) for each gramm of cell paste

Mechanical lysis

</center> The metho to disrupt the cells depends on the amount of biomass.

Mechanical lysis of the (shaking flask) cultivation

The metho to disrupt the cells depends on the amount of biomass.

• Sonification of the re-suspended pellet on ice
  • cycle number depends on the volume of the re-suspended cells (e.g. 3 mL means 3 cycles)
  • one cycle means sonification treatment for 1,5 min with Sonifier 450 by Branson, max. 50 %, cooled on ice, make sure not to heat the cells too much

Mechanical lysis of the (bio-reactor) cultivation

Cell disruption with a high-pressure homogenizer:

• high-pressure homogenisation with a Rannie Homogenizer:
• disruption of the cells by 3 cycles with cooling phases between the cycles, pressure = 1200 bar, make sure not to heat the cells too much

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)