Figure 2

Use of pC-DEP^N plasmid for making untagged or N-terminal GST-fusion proteins. Schematic of pC-DEP^N plasmid with the following features labeled: URA3 ORF in blue, f1 ori in white, CUP1 yeast promoters in green, glutathione S-transferase gene (GST) in yellow, the CYC1 yeast transcription terminator in red, the pBM1 origin of replication in white, and the bla gene conferring ampicillin resistance in grey. Unique restriction sites are labeled. Nucleotide sequence of the translation initiation region and corresponding protein translation sequence of the pC-DEP^N plasmid. The unique SacII site allows for genes of interest to be recombined upstream of the GST coding sequence. Demonstration of the function of pC-DEP^N plasmid. Left panel: Western blot analysis using α-GST antibodies of yeast cultures expression the indicated residues of the Hua1 gene that were recombined into the pC-DEP^N plasmid. Right panel: Coomassie Brilliant Blue stained SDS-PAGE gel of the same GST-fusion proteins as in left panel purified from BL21 (DE3) cells using GSH-agarose.