Title Generation of Expression Vectors for High-Throughput Functional Analysis of Target Genes in Schizosaccharomyces pombe
Author Jiwon Ahn1,2, Chung-Hae Choi1, Chang-Mo Kang3, Chun-Ho Kim3, Hee-Moon Park2, Kyung-Bin Song4, Kwang-Lae Hoe1, Misun Won1*, and Kyung-Sook Chung1*
Address 1Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea, 2Department of Microbiology, Chungnam National University, Daejeon 305-764, Republic of Korea, 3Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706, Republic of Korea, 4Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea
Bibliography Journal of Microbiology, 47(6),789-795, 2009,
Key Words expression vector, EGFP, DsRed, HA, gateway system, fission yeast
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, ura4+ and Saccharomyces cerevisiae LEU2 complementing leu1. These vectors contain 3 different strengths of the inducible promoter nmt1, 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.