We tested the feasibility of these vectors using the human gene G3BP1, the binding protein 1 of GTPase activating protein (SH3 domain) gene. At first, G3BP1 was cloned in the entry vector to be transferred to various destination vectors of Sz. pombe. The LR reaction combined the entry vector with each of the destination vectors pDES4X (no tags), pDES174 (3×HA), pDES176 (EGFP) and pDES6X (DsRed) (Figure 4A). After introducing each vector into Sz. pombe, we used immunoblot analysis to detect the expression of G3BP1 protein and the expression of N-terminus tagged G3BP1 using antibodies against G3BP1, HA, EGFP and Ds-Red. In all constructs, G3BP1 and N-terminal tagged G3BP1 proteins were expressed successfully (Figure 4B). In addition, human cDNA clone SCC112, a novel nuclear cell cycle regulatory protein gene, was transferred via an entry vector to pDES178–SCC112 by the Gateway LR reaction (Figure 4A). The subcellular localizations of G3BP1 (Ozeki et al., 2005) and SCC112 (Zheng et al., 2008), which have been previously reported, were investigated in Sz. pombe. After 12 h induction of the cloned protein, localization was visualized under fluorescence microscopy (Figure 4C). Our results confirmed those previously described; the fusion protein EGFP–G3BP1 was localized to the cytoplasm, showing a spot-like pattern, whereas EGFP–SCC112 was localized to the nucleus. As a control, GFP fluorescence in cells expressing EGFP alone was distributed throughout both the nuclear and cytoplasmic compartments. For comparison with the auxotrophic marker vector, the Sz. pombe hsp9+ gene was cloned into the vectors pDES175N carrying the LEU2 marker (Ahn et al., 2009) and pDES176 carrying the NAT marker, and the localization of the GFP–hsp9 fusion protein expressed from these vectors, pDES175N–hsp9+ and pDES176–hsp9+, was compared. In agreement with our previous study (Ahn et al., 2012), we observed that GFP–hsp9+ fusion proteins expressed from pDES175N–hsp9+ and pDES176–hsp9+ were localized in the nucleus after heat shock, indicating that these vectors are valuable tools for the production of heterologous proteins, similar to auxotrophic marker vectors in Sz. pombe.
Figure 4. Validation of the vectors in Sz. pombe. (A) Practical application of Gateway vectors to test correct fusion. An entry clone containing human G3BP1 was used to generate expression constructs that were either untagged or tagged with HA, EGFP or DsRed. Additionally, an entry clone containing human SCC112 was used to generate GFP-tagged expression constructs to test the correct localization of the fusion protein. (B) Validation of protein tagging by immunoblot analysis, using 30 µg protein in SDS–PAGE. Immunoblot analysis detected appropriately sized fusion proteins with anti-G3BP1, anti-HA, anti-RFP and anti-GFP. (C) The Gateway-generated GFP-tagged proteins localized properly in accordance with previous reports: EGFP–G3BP1 in the cytoplasm and EGFP–SCC112 in the nucleus. As a control, EGFP alone was distributed throughout in both cytoplasm and nucleus. (D) The subcellular localization of GFP–hsp9+ fusion proteins expressed from pDES175N (LEU2 marker, GFP tag) and pDES176 (NAT marker, GFP tag) carrying the hsp9+ gene
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Here, we report the construction of episomal vectors carrying the drug-resistance marker, natMX4, with three different strengths of the nmt1 promoter in minimal medium. We also generated expression vectors containing a destination cassette, which is suitable for high-throughput cloning of target genes using the Gateway system. These vectors were also modified to express target proteins with various tags at the N-terminus, which can be used for affinity binding and functional analysis of target genes. The use of drug-resistance markers in vectors does not limit parental strains with auxotrophic mutations, and the strain can be available for use in clone selection, whereas vectors with auxotrophic markers are at a disadvantage because they function only in strains harbouring the corresponding auxotrophic mutation. Moreover, vectors containing the nourseothricin-resistant marker, natMX4, are particularly advantageous when used with other vectors with auxotrophic markers, because only nourseothricin is effective in minimal medium and can therefore be used for novel experiments involving Sz. pombe. Furthermore, owing to the fact that the entire set of protein-coding open reading frames (ORFeome) of Sz. pombe was generated using a recombination-based cloning system, the genes in these ORFeome clones can be transferred to the destination vector in this study by the Gateway LR reaction (Matsuyama et al., 2006). Therefore, vectors containing a dominant selection marker, natMX4, may contribute to genome-wide investigation of the cellular mechanisms underlying the functions of multiple heterologous genes and help exploit the experimental convenience of Sz. pombe as a model organism.