We have constructed a series of galactose-inducible centromere-based yeast expression vectors to take advantage of the ability to quickly exchange cloned genes between vectors using Gateway® technology (Invitrogen, Carlsbad, CA, USA). Unlike pYES-DEST52 (Invitrogen) and other yeast vectors (1) that contain the 2-µm origin of replication, these vectors are based on CEN6 for plasmid maintenance because we observed the loss of 2 µm-based plasmids when expressing genes that are lethal to the cell (2). These eight plasmids complement the CEN-based vectors recently constructed by Van Mullem et al. (3) and add significantly to the repertoire of CEN-based yeast plasmids available for use with Gateway technology.
Two sets of constructs were created ((Figure 1)) based on previously tested galactose-inducible plasmids (4). Constructs in both sets contain an ampicillin resistance cassette, a chloramphenicol resistance cassette for negative selection of unrecombined vector, an f1 origin, CEN6, ARSH4 replicon, GAL1 promoter, and an auxotrophic marker (HIS3, TRP1, LEU2, or URA3). The four constructs in the first set ((Figure 1)A, one for each auxotrophic marker) are based on the previously published pJG485 (2) and contain a V5 epitope (5) and a 6×His tag as a C-terminal fusion to the expressed protein. The four analogous constructs in the second set ((Figure 1)B) lack the V5 epitope and the 6×His tag. These four constructs are similar to those constructed by Funk et al. (1) and Van Mullem et al. (3) except that each contains additional restriction sites before and after the Gateway recombination sites. These restriction sites in combination with the Gateway insert allow for the insertion of genes or gene fragments for creating fusion proteins in combination with the Gateway insert. Likewise, these sites may be used to clone additional epitope tags N-terminal or C-terminal to a gene of interest.Figure 1.
The constructs were made as described in the supplementary material available at the BioTechniques’ web site at www.BioTechniques.com/March2005/GeiserSupplementary.html . Vectors were confirmed by sequencing and by the ability of the vector to transform yeast on appropriate minimal medium; GenBank® accession numbers are described in (Table 1). The utility of plasmid pJG485 has been previously tested using YopO, which is lethal in yeast. This work is described in Nejedlik et al. (2) and confirms that the expected fusion protein is expressed in a GAL-regulated manner. Because the remaining vectors in the first set differ only in the auxotrophic marker present, we expect that vectors pJG518, pJG516, and pJG484 would behave equivalently in terms of cloning efficiency and protein expression. To confirm the utility of the second set of plasmids, we cloned YopO into pJG482. This construct is lethal in yeast in a manner similar to that observed with YopO in pJG485 (data not shown). Plasmids pJG515, pJG514, and pJG483 differ only in the auxotrophic marker present and are expected to behave equivalently.Table 1. GenBank Accession Numbers for Plasmids Described
This work was supported by grants to J.R.G. from Western Michigan University. A debt of gratitude is owed to Laura Nejedlik and Bruce Bejcek for discussions that led to this work.
The author declares no competing interests.