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Title 

Generation of expression vectors for high-throughput functional analysis of target genes in Schizosacchromyces pombe

 

분열효모를 이용한 타겟유전자의 초고속기능분석을 위한 벡터시스템 개발

Authors 

Ji Won AhnChung-Hae ChoiC M KangC H KimH M ParkK B SongKwang Lae HoeMi Sun WonKyung Sook Chung

Publisher 

Microbiological Society of Korea

Issue Date 

2009

Citation 

Journal of Microbiology, vol. 47, no. 6, pp. 789-795

Keywords 

DsRedEGFPExpression vectorFission yeastGateway systemHA

Abstract 

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4+ and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

ISSN 

1225-8873

Link 

http://dx.doi.org/10.1007/s12275-009-0010-4

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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