Team:Bielefeld-Germany/Production

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Revision as of 20:23, 22 June 2012

Production Protocols: These are the protocols for the cultivation and the downstream processing.

Cultivation

Expression of Laccase genes in E. coli

• Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]
• Medium: LB medium supplemented with 20 mg L^-1 chloramphenicol or autoinduction medium
  • Cultivations in LB-medium were supplemented with 0.1 % L-rhamnose as inducer, when the designated OD₆₀₀ was reached.
  • Autoinduction medium for expressing was supplemented with 1 mM IPTG.
• 150 mL culture in 500 mL shaking flask with baffles (Schott) with silicon plugs
• Cultivation temperature: 37 °C at 120 rpm

Expression of Laccase genes in P. Pastoris

• Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]
• Medium: LB medium supplemented with 20 mg L^-1 chloramphenicol or autoinduction medium
  • Cultivations in LB-medium were supplemented with 0.1 % L-rhamnose as inducer, when the designated OD₆₀₀ was reached.
  • Autoinduction medium for expressing was supplemented with 1 mM IPTG.
• 150 mL culture in 500 mL shaking flask with baffles (Schott) with silicon plugs
• Cultivation temperature: 37 °C at 120 rpm

Purification methods

Enzymatic cell lysis with lysozyme

• After cultivation biomass was collected by centrifugation at 5,000 g at 4 °C for 20 min.
• 1 g of biomass (wet weight) was suspended in 10 mL of enzyme buffer containing 0.1 % Triton X-100, 2 µL benzonase (250 U/µL) and 40 µL of lysozyme (100 mg mL^-1)
• Incubation for 30 min at 4 °C
• reaction mixture was centrifuged for 30 - 90 min at 15,000 g at 4 °C

Release of periplasmic protein fraction from E. coli by cold osmotic shock

Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].

• Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells.
• Discard the entire supernatant.
• Resuspend the cells ice-cold cell fractionating buffer 1. The resulting volume should be 1/4 of the former suspension volume.
• Incubate for 20 min on ice. Ivert the suspension at regular intervals to counteract sedimentation.
• Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
• Discard the entire supernatant.
• Resuspend the cells ice-cold cell fractionating buffer 2. The resulting volume should be 1/4 of the former suspension volume.
• Incubate for 10 min on ice under regular invertion.
• Centrifuge the cell suspension for 15 min at 14,000 g (4 °C).
• Save the supernatant, which contains the periplasmatic proteins.
• If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter.

Inclusion body clean-up

• harvest the cells by centrifugation (30 min, 10,000 g, 4 °C)
• resuspend pellet and disrupt cells
• centrifuge lysate (60 - 90 min, >17000 g, 4 °C)
• wash pellet at least two times with water to remove water-soluble proteins
• after washing the pellet: incubate the pellet in denaturation buffer for 60 min, 4 °C with vertical rotator
  • final concentration in denaturation buffer: 0.5 mg wet biomass per mL
• centrifuge (60 min, >17,000 g, 4 °C)
  • the higher the speed, the better the result
• collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
• centrifuge (60 min, >17,000 g, 4 °C)
• collect supernatant and discard pellet

Ammonium sulfate precipitation

• Mix fraction you want to clean-up with ammonium sulfate
  • To precipitate S-layer proteins from Corynebacterium, 40 % ammonium sulfate saturation concentration is a good concentration (247 g L^-1 ammonium sulfate at 25 °C)
• Incubate 30 min at room temperature on a shaker
• Centrifuge (the faster and longer the better) and solve the precipitate in water or buffer

Ultra-/Diafiltration

• Arrange the filtration module as shown on the right side.
• Microfiltration (0.22 µm) or cross flow filtration with 300 kDa (we used a Milipore Pellicon XL 300) membrane of sample before ultrafiltration.
• For concentrating the sample just filter it until the desired volume is left in the feed reservoir. For diafiltration (e.g. buffer exchange, desalting) dilute the feed reservoir several times and filter continously.
• Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
  • 50 or 100 kDa cut-off
  • 50 cm² filtration area
  • tangential flow filter
  • Hydrophilic polyvinylidene fluoride membrane
• Used pump: SciLog TANDEM 1081 peristaltic pump
  • flow rate during filtration: 40 mL min^-1

Ion exchange chromatography (IEX) for S-layer proteins from Corynebacterium

• used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
• flow rate: 1 mL min^-1
• equilibrate column with > 10 column volumes of binding buffer
• inject sample and wash column with binding buffer until UV signal is constant
• elute with 20 %, 40 % and 60 % of binding / elution buffer mix and collect fractions
• elute remaining proteins with 100 % elution buffer

Ion exchange chromatography (IEX) for S-layer proteins from Lysinibacillus sphaericus

• tested with <partinfo>K525405</partinfo>
• used column: DEAE HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
• flow rate: 0.5 mL min^-1
• equilibrate column with 20 column volumes of binding buffer
• inject sample and wash column with binding buffer until UV signal is constant
• elute with 10 % of binding / elution buffer mix and collect fraction
• elute remaining proteins with 100 % elution buffer

Hydrophobic Interaction Chromatography (HIC) for S-layer proteins

• tested with <partinfo>K525405</partinfo> and <partinfo>K525311</partinfo>
• used column: Butyl HiTrap 1 mL with [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus]
• flow rate: 0.5 mL min^-1
• equilibrate column with 20 column volumes of binding buffer
• inject sample and wash column with binding buffer
• elute in 10 % steps of binding / elution buffer mix and collect fractions
  • <partinfo>K525405</partinfo> elutes at 70 % buffer B
  • <partinfo>K525311</partinfo> elutes at 50 % buffer B
• elute remaining proteins with 100 % elution buffer

His-tag affinity chromatography

• Column: 1 mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare]
• Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
• Discard the supernatant and freeze bacterial pellet at -20 °C for at least 30 min
• Resuspend the pellet in 5 mL binding buffer for each gram of cell paste
• Wash column with 5 - 10 mL of deionized water
• Equilibrate column with 5 - 10 mL of binding buffer

denaturing

• For buffers see table buffers for his-tag affinity chromatography
• Mechanical lysis:
  • Sonification on ice for 6 - 10 min with Sonifier 450 by [http://www.gehealthcare.com/ Branson], max. 20 W, cooled on ice
• Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
• Centrifuge at 15,000 g for 30 min at 4 °C
• Filter sample (sterile filter or 300 kDa cut-off)

• Syringe method
  • Equilibrate with binding buffer
  • Load sample onto column
  • Wash with 10 mL binding buffer
  • Elute with 5 mL of elution buffer with increasing imidazole concentrations
  • Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
  • Re-equilibrate the column with binding buffer

• ÄKTA method
  • Equilibrate with 20 column volumes binding buffer, 0.5 mL min^-1
  • Load sample onto column
  • Wash with binding buffer until UV signal is constant
  • Elute with 50 mM imidazol
  • Elute remaining proteins with 500 mM imidazol

non-denaturing

• For buffers see table buffers for his-tag affinity chromatography
• Enzymatic lysis:
  • Add 0.2 mg L^-1 lysozyme, 3 units of Benzonase per mL of culture volume and 1 mM MgCl₂
  • Stirr for 30 in at 4 °C
• Centrifuge at 10,000 g for 30 min at 4 °C
• Load sample onto the column
• Wash with 10 mL binding buffer
• Elute with 5 mL of elution buffer with increasing imidazole concentrations
• Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
• Re-equilibrate the column with binding buffer

Production and purification strategies

Fusion proteins of SgsE and SbpA

• Cultivation
  • Bioreactor: Bioengineering NLF22 7 L or [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] with Bioengineering DCU
  • Medium: HSG medium with 20 mg L^-1 chloramphenicol or 100 mg L^-1
  • Culture volume: 2.5 - 5 L
  • Inoculation OD₆₀₀: 0.1 - 0.4
• DO: 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
• pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
• Antifoam: BASF pluronic PE-8100
• Induction after 4 h cultivation time with 0.2 % rhamnose and 1 mM IPTG (in culture medium)
• Harvest after 8 - 13 h

• Cell lysis
  • Centrifuge down the cells (10,000 g, 30 min, 4 °C)
  • Resuspend pellet in enzyme buffer or binding buffer for denaturing his-tag purification
  • Cell lysis with high-pressure homogenizer (800 bar, 3 cycles at 4 °C)
  • Centrifuge down the lysate (10,000 g, 60 min, 4 °C)

• Inclusion body clean-up
  • wash pellet from cell lysis with water twice
  • after washing the pellet: incubate the pellet in denaturation buffer for 60 min, 4 °C with vertical rotator
    • final concentration in denaturation buffer: 0.5 mg wet biomass per mL
  • centrifuge (60 min, >17,000 g, 4 °C)
    • in general with all centrifugations during this clean-up: the higher the speed, the better the result
  • collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
  • centrifuge (60 min, >17,000 g, 4 °C)
  • collect supernatant and discard pellet

• Filtration
  • Arrange the filtration module as shown on the right side.
  • Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
    • This step is for removing cell debris
  • Diafiltrate with 100 kDa membrane against denaturation buffer
    • constantly delute permeate with the buffer, keeping the permeate volume as low as possible
  • Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
    • 50, 100 or 300 kDa cut-off
    • 50 cm² filtration area
    • tangential flow filter
    • Hydrophilic polyvinylidene fluoride membrane
  • Used pump: SciLog TANDEM 1081 peristaltic pump
    • flow rate during filtration: 40 mL min^-1

OR WHEN USING A HIS-TAGGED PROTEIN (RECOMMENDED):

• His-6-affinity tag purification
  • For buffers see table buffers for his-tag affinity chromatography
  • Mechanical lysis as described above in binding buffer
  • Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
  • Centrifuge at 15,000 g for 30 min at 4 °C
  • Filter sample (sterile filter or 300 kDa cut-off)
  • Equilibrate column (1 mL HisTrap FF crude from GE Healthcare) with 20 column volumes binding buffer, 0.5 mL min^-1
  • Load sample onto column
  • Wash with binding buffer until UV signal is constant
  • Elute with 50 mM imidazol
  • Elute remaining proteins with 500 mM imidazol

• Dialysis
  • Fill retentate from DF/UF or chromatography elution fraction in dialysis tube ([http://www.carl-roth.de/ Roth], cellulose, 10 kDa cut-off)
  • Dialyse against ddH₂O for 18 h at 4 °C in the dark
  • After dialysis: centrifuge down the precipitation (45 min, 17,000 g, 4 °C) and collect the supernatant
  • Measure protein concentration in supernatant, dilute to 1 mg mL^-1 with ddH₂O and store at 4 °C in the dark

• Scheme of purification strategy for S-layer (fusion) proteins:

With inclusion body purification:

With HisTrap:

• Further purification of dirty inclusion body purification by IEX and HIC as described above

Fusion proteins of CspB without lipid anchor with TAT-sequence

• Cultivation
  • 150 mL culture in 500 mL or 300 mL culture in 1000 mL shaking flasks with baffles (Schott) with silicon plugs
  • Medium: autoinduction medium
  • Cultivation temperature: 37 °C at 120 rpm

• Supernatant precipitation
  • Centrifuge down the cells (10000 g, 30 min, 4 °C) and collect the supernatant
  • To precipitate S-layer proteins from Corynebacterium, i.e. 40 % ammonium sulfate saturation concentration (247 g L^-1 ammonium sulfate at 25 °C)
  • Incubate 30 min at room temperature on a shaker
  • Centrifuge (the faster and longer the better) and solve the precipitate in binding buffer for IEX

• Filtration
  • Arrange the filtration module as shown on the right side.
  • Collect permeate of cross flow filtration with 300 kDa membrane of sample before ultrafiltration
    • This step is for removing cell debris
  • Diafiltrate with 50 kDa membrane against binding buffer for IEX
    • constantly delute permeate with the buffer, keeping the permeate volume as low as possible
  • Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50], XL 100 or XL 300 membranes
    • 50, 100 or 300 kDa cut-off
    • 50 cm² filtration area
    • tangential flow filter
    • Hydrophilic polyvinylidene fluoride membrane
  • Used pump: SciLog TANDEM 1081 peristaltic pump
    • flow rate during filtration: 40 mL min^-1

• Ion exchange chromatography
  • used column: DEAE HiTrap 1 mL with [http://www.gehealthcare.com/ GE Healthcare] ÄKTA
  • flow rate: 1 mL min^-1
  • equilibrate column with 10 column volumes of [Team:Bielefeld-Germany/Protocols#Buffers_for_S-layer_IEX | binding buffer]
  • inject sample and wash column with binding buffer
  • elute with 20 %, 40 % and 60 % binding / elution buffer mix and collect samples -> take the cleanest fraction for further work
  • elute remaining proteins with 100 % elution buffer

• Scheme of purification strategy for CspB S-layer (fusion) proteins:

Recrystallization of S-layer proteins

Recrystallization of SgsE and SbpA in solution

• after purification: dialyse 18 h at 4 °C against ddH₂O in the dark
• centrifuge and use supernatant -> this is the monomeric protein solution
• dialyse 18 h at 4 °C against
  • HBSS pH 7.4 for SgsE
  • recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl₂) for SbpA

Immobilization of SgsE on silica beads

• after purification: dialyse 18 h at 4 °C against ddH₂O in the dark
• centrifuge and use supernatant -> this is the monomeric protein solution
• measure protein concentration
• dilute purified monomeric S-layer solution to 1 mg mL^-1 protein with ddH₂O -> store in the dark at 4 °C
• suspend silicium dioxide beads in HBSS (pH 7.4) and mix it with the 1 mg mL^-1 S-layer solution
  • ratio of beads to protein can be varied
  • 0.1 mg mL^-1 final protein concentration
  • contact with HBSS buffer will start assembly of SgsE
• incubate on vertical rotator at room temperature for 4 h
• after incubation: centrifuge down the beads (1 min, > 15000 g), wash them twice with ddH₂O and store them afterwards in ddH₂O at 4 °C in the dark

Immobilization of SbpA on silica beads

• after purification: dialyse 18 h at 4 °C against ddH₂O in the dark
• centrifuge and take supernatant -> this is the monomeric protein solution
• measure protein concentration
• dilute purified monomeric S-layer solution to 1 mg mL^-1 protein with ddH₂O -> store in the dark at 4 °C
• suspend silicium dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl₂) and mix it with the 1 mg mL^-1 S-layer solution
  • ratio of beads to protein can be varied
  • 0.1 mg mL^-1 final protein concentration
  • contact with recrystallization buffer will start assembly of SbpA
• incubate on vertical rotator at room temperature for 4 h
• after incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH₂O and store them afterwards in ddH₂O at 4 °C in the dark

Immobilization of SgsE on silicon wavers

• used wavers: p-type / bor
• wash wavers with 70 % ethanol and ddH₂O
• cover the waver with 0.1 mg mL^-1 purified protein in HBSS buffer for 4 h

Immobilization of SbpA on silicon wavers

• used wavers: p-type / bor
• wash wavers with 70 % ethanol and ddH₂O
• cover the waver with 0.1 mg mL^-1 purified protein in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl₂) for 4 h