"

From 2012.igem.org

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

Contents 1 Cultivation 1.1 Expression of Laccase genes in E. coli 1.2 Expression of Laccase genes in P. Pastoris 2 Purification methods 2.1 Enzymatic cell lysis with lysozyme 2.2 His-tag affinity chromatography

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: Pichia Pastoris
• Medium:
• 200 mL culture in 1000 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

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

• 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)

• 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