Montsrrat Zieger, Q Biogene.

Overview

  • Keywords: Protein extraction, gram-positive bacteria, restriction enzyme.
  • Aim of the study: Demonstrate the capacity of the FastPrep® System to deal with otherwise difficult to lyse bacteria.
  • Application: Restriction enzymes extraction
  • Sample type: Bacillus amyloliquefaciens and Staphylococcus aureus 3A
  • Material: FastPrep-24™ instrument, FastProtein™ Blue Kit containing Lysing Matrix B tubes

Protocols & Parameters

  1. Cell density.

    • Sonication: Bacterial suspensions of 0.2 g wet weight (w/w) and 0.15 g (w/w) per ml of buffer for Bacillus amyloliquefaciens and Staphylococcus aureus 3A, respectively.
    • FastPrep®: Bacterial suspensions of 0.1 g (w/w) and 0.4 g (w/w) per ml and 0.15 g (w/w) per ml of buffer for Bacillus amyloliquefaciens and Staphylococcus aureus 3A, respectively.

  2. Disruption.

    • Sonication: Bacteria are disrupted at 50% maximum intensity (large tip) for Bacillus amyloliquefaciens and 20% maximum intensity (small tip) for Staphylococcus aureus 3A with a Branson Sonicator B30. Temperature is maintained at 4°– 5°C by cooling in an ice salt water bath. Sonication was continued for 10 min in 40 sec. bursts for Bacillus amyloliquefaciens and 60 sec. in 5 sec. bursts for Staphylococcus aureus 3A.
    • FastPrep®: The FastProtein™ Blue matrix was used. Tubes containing the lysing matrix and samples were prechilled at 4°C then mixed. Samples are homogenized with the FastPrep-24™ instrument at speed 6.0 for 40 sec. for Bacillus amyloliquefaciens and at speeds 4.0 and 6.0 for 20 sec. and 40 sec. respectively for Staphylococcus aureus 3A. The tubes were returned to the ice bath. Homogenization and chilling was repeated for all time points. At each time point a 50 μl sample was taken, centrifuged for 5 min at 4°C in a benchtop centrifuge and tested for OD260 and activity.

Results


Lanes 1 to 8 correspond to the samples processed in the FastPrep-24™: 1 to 4 are at 0.4 mg/ml and 5 to 8 at 0.1 mg/ml. 1 and 5 at time 0, 2 and 6 at 40 sec., 3 and 7 at 2 x 40 sec., 4 and 8 are 3 x 40 sec. Lanes 9, 10 and 11 correspond to sonication samples (S) taken at 4 x 40 sec., 7 x 40 sec. and 9 x 40 sec., respectively. Lane 12:λ DNA cut by purifi ed Bam HI (C).

Conclusion

These experiments clearly show that the FastPrep-24™ instrument using FastProtein™ Blue matrix can be used to successfully extract unstable enzymes from gram positive bacteria. Even in cases where sonication can release active materials (such as the Bacillus amyloliquefaciens experiments here), the lysing time can be reduced by approximately 60%. For samples like Staphylococcus aureus 3A that require longer and less efficient methods of lysis (such as French Press), the FastPrep® system offers clear advantages for extraction of active proteins.