Generation of expression vectors for high-throughput functional analysis of target genes in Schizosaccharomyces pombe.
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 (3x 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.
Ahn
Jiwon
J
Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea.
Choi
Chung-Hae
CH
Kang
Chang-Mo
CM
Kim
Chun-Ho
CH
Park
Hee-Moon
HM
Song
Kyung-Bin
KB
Hoe
Kwang-Lae
KL
Won
Misun
M
Chung
Kyung-Sook
KS
D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D008967:Molecular Biology / Q000379:methods*; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins