Abstract
Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology.
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Acknowledgements
This project was supported by Betatech (France), the French government supported ANR Debaciem and the European FP7 Project (Electroextraction, FP7-SME-2007-1, grant 222220). Research was conducted in the scope of the EBAM European Associated Laboratory (LEA). This research was partially conducted in the framework of COST Action TD1104. Thanks are due to Pr. Laurent Paquereau and Laurence Nieto (IPBS) for stimulating discussions and to Dr. Elisabeth Bellard and Ms. Caroline Ladurantie for their technical expertise.
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Coustets, M., Al-Karablieh, N., Thomsen, C. et al. Flow Process for Electroextraction of Total Proteins from Microalgae. J Membrane Biol 246, 751–760 (2013). https://doi.org/10.1007/s00232-013-9542-y
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DOI: https://doi.org/10.1007/s00232-013-9542-y