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Journal of Biochemistry Advance Access originally published online on February 23, 2009
Journal of Biochemistry 2009 145(6):701-708; doi:10.1093/jb/mvp028
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

A Simple and Immediate Method for Simultaneously Evaluating Expression Level and Plasmid Maintenance in Yeast

Jun Ishii1, Keiko Izawa2, Shizuka Matsumura2, Kanako Wakamura2, Takanori Tanino2, Tsutomu Tanaka1, Chiaki Ogino2, Hideki Fukuda1 and Akihiko Kondo2,*

1Organization of Advanced Science and Technology, Kobe University, Japan; and 2Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan

*To whom correspondence should be addressed. Tel: +81-78-803-6196, Fax: +81-78-803-6196, E-mail: akondo{at}kobe-u.ac.jp

Received January 6, 2009; Accepted February 12, 2009


  Abstract

To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.

Key Words: yeast, expression system, plasmid maintenance, green fluorescent protein, flow cytometry

Abbreviations: CEN/ARS, centromere-autonomously replicating sequence; EGFP, enhanced green fluorescent protein; FCM, flow cytometry; GFP, green fluorescent protein; IR, inverted repeat; MCS, multiple cloning site; PGK5', PGK1 promoter; PGK3', PGK1 terminator; SD, synthetic dextrose; YPDA, yeast extract-peptone-dextrose-adenine


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