Pulsed electric field (PEF)-assisted protein recovery from Chlorella vulgaris is mediated by an enzymatic process after cell death

Pulsed electric field (PEF) treatment has so far been considered as ineffective for protein extraction, but the parameters that influence protein extraction efficiency have not been identified fully. Using Chlorella vulgaris in this study, we show that up to half of the proteins present in the cell can be extracted via PEF treatment and an incubation step for up to 24 h. The extraction efficiency is highly influenced by biomass concentration during the incubation step and the incubation temperature: in our experiments we were able to extract half of the proteins present in the cells at dilute concentrations (5 mg.ml(-1)), but this efficiency decreases with increasing biomass concentration, indicating that diffusion gradients play a role in the protein release. However, we also show that not only diffusion plays a role, but also a biological process that requires suitable conditions. We observed that PEF treated cells undergo programmed cell death, indicated by DNA laddering. Furthermore cold and hot temperatures as well as protease inhibition impair the release of proteins. In Western blots we can observe that some proteins are not released when the activity of proteases is blocked. We therefore conclude that protein release is facilitated by an enzyme-driven process that occurs after PEF-triggered cell death, and that this process is prone to extreme conditions.