Production

Here all our methods according to cultivation and purification are listed.

Pre-Cultivation

Precultivation of E.Coli KRX (with or without BioBrick)

• 50 mL LB-medium, if nessassary with 20-60 mg L-1 chloramphenicol or with 100-300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
• 1 mL Glycerinculture or 1 colony
• Cultivation temperature: 37 °C
• Shaking at 140 rpm

Precultivation of E.Coli Rosetta Gami 2 (with or without BioBrick)

• 50 mL LB-medium with 60 mg L-1 chloramphenicol and with 300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
• 1 mL Glycerinculture or 1 colony
• Cultivation temperature: 37 °C
• Shaking at 140 rpm

Precultivation of Pichia Pastoris GS115 (complex-medium)

• 50 mL YPD-medium in 300 mL shaking flask with baffles (Schott) with silicon plugs
• 1 mL Glycerinculture or 1 colony
• Cultivation temperature: 30 °C
• Shaking at 140 rpm

Precultivation of Pichia Pastoris GS115 (minimal-medium)

• 50 mL YNB-medium in 300 mL shaking flask with baffles (Schott) with silicon plugs
• 1 mL Glycerinculture or 1 colony
• Cultivation temperature: 30 °C
• Shaking at 140 rpm

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 special buffer or binding buffer for each gramm of cell paste

Solubilization of inclusion bodies

• centrifugation of the celllysate at 40,000 g for 30 minutes
• resuspend the pellet of the lysate in 1 mL 6M Urea solution, incubation for 1 hour
• centrifugation for 10 minutes at 10,000 rpm
• resuspend the pellet in SDS running buffer

Cell disruption strategies

B-PER lysis (chemical lysis)

B-Per bacterial Protein Extraction Reagnt was used for a cell disruption screening according to the following protocol of Thermo Scientific.

• add 4 mL B-Per Reagent per gram of cell pellet
• resuspend the cell pellet by pipetting the suspension up and down until it is homogenous.
• incubate the solution for 10- 15 min at room temperature
• centrifuge lysate at 15.000g for 10 min
• decant the supernatant in a clean tube

The lysate is ready for a following purification step.

enzymatical lysis with lysozym

The lysis with lysozym was used for a cell disruption screening. The following protocol was utilized:

• resuspend the cell pellet in 600 µL of lysozym-solution per gramm
• incubate the solution for 1 h at 4°C
• centrifuge the lysate toseperate soluble proteins from insoluble proteins and cell debris.

combination of chemical and enzymatical lysis

B-Per bacterial Protein Extraction Reagnt was used for a cell disruption screening according to the following protocol of Thermo Scientific.

• add 4 mL B-Per Reagent per gram of cell pellet
• add 2µL of lysozym-solution(50 mg mL ^-1) and 2 µL DNaseI (2500 U mL ^-1) per mL B-Per Reagent. (For laccases:Do not use EDTA!)
• resuspend the cell pellet by pipetting the suspension up and down until it is homogenous.
• incubate the solution for 10- 15 min at room temperature
• centrifuge lysate at 15.000g for 10 min
• decant the supernatant in a clean tube

The lysate is ready for a following purification step.

Mechanical lysis

The method of choice to disrupt the cells depends on the amount of biomass.

Mechanical lysis of the (shaking flask) cultivation

Sonication

• Sonication of the re-suspended pellet on ice
  • cycle number depends on the volume of the resuspended 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

Precellyse 24 homogenization

• homogenization with the Precellyse 24
  • fill the precellyse tubes with a sample volume between 1 mL up to 1,5 mL (for 2 mL tubes)
  • homogenize the samples for 3 cycles (6500 rpm for 35 sec. ), to make sure not to heat the cells to much, the sample were stored for 5 min in ice between 2 cycles.

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

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)

An typical chromatogram of purified laccases is illustrated in the following grafic:

A typical chromatogram of the purification procedure of laccases. the different steps of the purification are illustrated: 1.) Equlibration, 2.) Sample Loading, 3.) Wasch, 4.)Elution, 5.) Regeneration