Journal of Microbiology

Validation Study

Generation of Expression Vectors for High-Throughput Functional Analysis of Target Genes in Schizosaccharomyces pombe: Jiwon Ahn , Chung-Hae Choi , Chang-Mo Kang , Chun-Ho Kim , Hee-Moon Park , Kyung-Bin Song , Kwang-Lae Hoe , Misun Won , Kyung-Sook Chung; J. Microbiol. 2009;47(6):789-795. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0010-4

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.

Expression of human hypoxanthine phosphoribosyltransferase in Insect Cells Using a Baculovirus Vector: Lee, Chong Ho , Yang, Ji Won , Baek, Sang Ki , Yang, jai Myung; J. Microbiol. 1995;33(1):85-90.

Abstract: The hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to the mononucleotide, IMP and GMP, respectively. For construction of recombinant AcNPV carrying human HPRT, a transfer vector p918 constructed by cloning full-length cDNA for human HPRT into pVL1393 and AnNPV genomic DNA were co-transfected into Sf21 cells. The tissue culture fluid containing extracellular virus was plaque assayed and a recombinant virus with occlusion minus phenotype was obtained by three rounds of plaque purification. Southern blot analysis and PRC results confirmed the insertion of the human HPRT cDNA within the recombinant virus(AcHPRT918). SDS-PAGE and Western blot analysis of the Sf21 cell extracts infected with AcHPRT918 indicated that human HPRT was expressed in insect cell. Large quantities of functional HPRT expressed in insect cells would facilitate characterization of the biological properties of this enzyme.

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