The plasmid pSpot5 is an expression vector for Spot-Tag® fusion proteins in S. cerevisiae. After cloning the protein of interest (POI) into the multiple cloning site (MCS) provided by pSpot5, the Spot-Tag® (sequence: PDRVRAVSHWSS) will be fused to the N-terminus of the POI.
Transform a suitable leu2 deletion strain of S. cerevisiae using pSpot5 and standard methods and grow on media lacking leucine. Protein expression is constitutive under the strong triosephosphate isomerase (TPI1) promoter. The resulting Spot-Tag® fusion protein can be purified using the ChromoTek Spot-Trap®.
Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. The vector confers resistance to ampicillin (100 μg/ml) to E. coli hosts. (This plasmid was designed and generated by Dr. Philipp Kaiser from the NMI.)