The Use of Pulsed Electric Fields for Protein Extraction from Nanochloropsis and Chlorella
Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on batch and flow processes electro extraction on yeasts proved that pulsed electric field technology was highly effective and preserve the activity of released cytosolic proteins. As induction of electro permeabilization is under the control of target vesicle size, protocols were chosen to affect only the plasma membrane and not vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris) Due to the size selective effect of electro permeabilization, as the mean diameter for N. salina is only 2.5 mm, we used stronger field strengths than for C. Vulgaris. A train of repetitive 2 ms long pulses of alternating polarities was delivered. The electric treatment was followed by a 24 h incubation period in a salty buffer containing DTT at room temperature. The amount of total proteins release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS PAGE. Similar results were obtained with C. vulgaris and N. Salina under optimized electrical conditions.