Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies
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The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
KeywordsChannelrhodopsin-2 Electrorotation Dielectrophoresis Giant protoplast Cell wall digestion Patch clamp
We thank C. Bamann (MPI of Biophysics) for generously providing us with the P. pastoris ChR2(C128T)YFP strain, M. Heidbreder (University of Würzburg) for support with LSM imaging, A. Pustlauck and E. Kaindl (MPI of Biophysics) for technical support and J. Reichert (MPI of Biophysics), A. Gessner and M. Behringer (University of Würzburg) for construction of electrofusion chambers.
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