The Journal of Membrane Biology

, Volume 245, Issue 12, pp 815–826

Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies

  • Ulrich Terpitz
  • Sebastian Letschert
  • Ulrich Bonda
  • Christoph Spahn
  • Chonglin Guan
  • Markus Sauer
  • Ulrich Zimmermann
  • Ernst Bamberg
  • Dirk Zimmermann
  • Vladimir L. Sukhorukov
Article

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.

Keywords

Channelrhodopsin-2 Electrorotation Dielectrophoresis Giant protoplast Cell wall digestion Patch clamp 

Supplementary material

232_2012_9484_MOESM1_ESM.docx (3.7 mb)
Supplementary material 1 (DOCX 3744 kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ulrich Terpitz
    • 1
    • 2
  • Sebastian Letschert
    • 2
  • Ulrich Bonda
    • 2
  • Christoph Spahn
    • 2
  • Chonglin Guan
    • 2
  • Markus Sauer
    • 2
  • Ulrich Zimmermann
    • 4
  • Ernst Bamberg
    • 1
    • 3
  • Dirk Zimmermann
    • 3
  • Vladimir L. Sukhorukov
    • 2
  1. 1.Department of Biophysical ChemistryMax-Planck-Institute of BiophysicsFrankfurtGermany
  2. 2.Department of Biotechnology and BiophysicsJulius-Maximilians-University WürzburgWürzburgGermany
  3. 3.Department of Biochemistry, Chemistry and PharmacyJohann-Wolfgang-Goethe University FrankfurtFrankfurtGermany
  4. 4.ZIM Plant TechnologyHennigsdorfGermany

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