ArticleThe Journal of membrane biology2012
Dielectric analysis and multi-cell electrofusion of the yeast Pichia pastoris for electrophysiological studies.
Article in The Journal of membrane biology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Investigation of Zebrafish Embryo Membranes at Epiboly Stage through Electrorotation Technique.Membranes · 2023Article
- Expansion Microscopy for Cell Biology Analysis in Fungi.Frontiers in microbiology · 2020Article
- Cell Electrofusion in Centrifuged Erythrocyte Pellets Assessed by Dielectric Spectroscopy.The Journal of membrane biology · 2016Article
- The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination.Scientific reports · 2015Article
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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