Dr. Loїc Faye, ANGANY Genetics, France.

Overview

  • Keywords: Protein expression, plant screening, recombinant protein
  • Aim of the study: Selection of the best producers of a recombinant protein used for allergic patients desensitization
  • Application: Western Blot analysis
  • Sample type: Leaves
  • Material: FastPrep-24™ instrument, 2 ml Lysing Matrix D tubes containing 1.4 mm ceramic spheres
  • Buffers: Laemmli denaturing sample buffer containing 60 mM Tris-Cl pH 6.8, 1% SDS, 10% glycerol, 2% beta-mercaptoethanol

Protocols & Parameters

  1. Snap one tobacco leaf disc (10 mg) in a Lysing Matrix D tube
  2. Add 200 μl of denaturing buffer to the Lysing Matrix D tube
  3. Homogenize the plant tissue with the FastPrep-24™ instrument for 60 seconds at a speed setting of 4.0 m/s
  4. Transfer supernatant from the Lysing Matrix D tube into an Eppendorf tube
  5. Boil each sample for 5 min and then centrifuge 1 min at 12.000 rpm to pellet the cellular debris
  6. Load 15 μl of each supernatant in a 15% polyacrylamide gel for protein separation by electrophoresis

After SDS-PAGE proteins are either stained in the gel or transferred on a membrane for Western Blot analysis.

Results

Screening for expression of a recombinant allergen from house dust mite in tobacco plants using the FastPrep® instrument for sample preparation.

Each lane in the upper panel shows the protein pattern (SDS-PAGE) obtained for one leaf disc cut out from 12 different tobacco plants used for transient expression of the recombinant allergen. The lower panel is a Western Blot used for identification of the best allergen producers.

The same samples were used in the upper and lower panels. This procedure is part of the AllergoPur™ production platform developed in ANGANY Genetics, France.

Conclusion

  • The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis(SDS-PAGE) and Western Blot analysis.
  • The FastPrep-24™ instrument has also been successfully used for protein extraction from other plants (Arabidopsis,
    lettuce etc...) in the same conditions using less than 15 mg of plant material for sample preparation.
  • Similar experiments have been performed for protein extraction in non-denaturing conditions using ice cold buffer
    (100mM Tris buffer, pH 7.4 containing 10% sucrose, 5 mM EDTA, 0.28% b-ercaptoethanol, 5 ml PMSF and 0.3 ml
    aprotinin) and plant samples stored at -70°C in lysing matrix D tubes before extraction.
  • The process is then scaled-up by using the BigPrep™ adapter for homogenization in 2 x 50 ml tubes. Two large
    protein samples are prepared simultaneously from 1-2 g of plant tissues in 50 ml lysing Matrix D tubes.