High-Throughput Liberation of Water-Soluble Yeast Content by Irreversible Electropermeation (HT-irEP)

¹ Biochemical Engineering, School of Engineering and Science, International University Bremen, Bremen, Germany.; Alfred Wegener Institute for Polar and Marine Research (AWI), Bremerhaven, Germany.
² Biochemical Engineering, School of Engineering and Science, International University Bremen, Bremen, Germany.
³ Faculty of Biology, University of Sofia, Sofia, Bulgaria.

^* To whom correspondence should be addressed. E-mail: maxim.zakhartsev{at}awi.de.

	Abstract

The article describes a high-throughput method for the liberation of water-soluble cell contents by exploiting the phenomenon of irreversible membrane electropermeation (HT-irEP). The method is exemplified in recombinant proteins and plasmid liberation from yeast Saccharomyces cerevisiae on the detectable level. Obtained extracts are pure enough to be readily applied for further analytical analysis such as enzyme assay, PCR, and so on. From the same HT-irEP extract, one can measure activity of the target protein and perform amplification of the corresponding gene from the DNA vector by PCR for recombinant protein with intracellular expression. Therefore, the method is suitable for the high-throughput screening (HTS) of yeast libraries where extracellular expression of recombinant protein is problematic. The method can be easily automated and integrated into existing HTS systems.

Key Words: high-throughput screening, yeast library, plasmid liberation, protein liberation, membrane electropermeation

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