Team:Bielefeld-Germany/Protocols/Production

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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


Used Chromatographycolumns:
left 1 mL HisTrap FF crude by GE Healthcare
middle 15 mL HisTrap FF crude by GE Healthcare
right TALON His-Tag Purification Resin by Clonetech
  • 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
Äkta Prime by GE Healthcare
  • 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)