Nondistractive extraction of intracellular molecules from yeast using PEF-assisted autolysis

This paper discusses the possibility of nondestructive extraction of intracellular proteins of yeast (Saccharomyces cerevisiae) by means of pulsed electric fields (PEFs) combined with autolysis. First, the effects of PEFs (20 kV/cm, 600 ns) on yeast and the subsequent cell wall collapse phenomenon were studied by morphological observation of fluorescence staining. Morphological observation shows that the PEFs increase the permeability of the cell membrane and also disrupt the intracellular vacuoles, whereas the cell wall morphology remains unchanged. However, the cell wall was collapse during the postPEF incubation. Next, the amount of extracted protein was evaluated by using the BCA assay. The amount of extracted protein in the PEF treated suspension was only a little, whereas the additional incubation promotes the extraction. Lastly, the size of the extracted proteins was investigated by using both Native-PAGE and SDS-PAGE. The PEF-treated and incubated suspension contains the large protein over 250 kDa.