Effects of TEV protease expression on wild-type and mcm7-GFPN760::Tcs S. pombe strains. A. Western analysis comparing levels of expression of TEV protease in strain P1292 in – thiamine medium (lane 2) and + thiamine medium (lane 3). Lane 1 is protein extract from a strain lacking the pREP3X-TEV-NLS plasmid (P138). B. Expression of TEV protease does not affect the viability of S. pombe. Serial dilutions of strain P1292 were spotted onto plus (+T) and minus (-T) medium and incubated at the temperatures shown. C. Design of Mcm7-GFPN760::Tcs. The Mcm7 gene was modified so that a TEV cleavage site, fused to GFP is expressed after the last codon of the Mcm7 reading frame. Cleavage by TEV protease liberates GFP with an N- terminal serine, which is stable according to the N-end rule. D. Western analysis of the mcm7-GFPN760::Tcs strains, using anti-GFP antibody. Lane 1: untagged control strain (P138); lane 2: mcm7-GFPN760::Tcs strain (P1288); lane 3: mcm7-GFPN760::Tcs containing pREP3X-TEV-NLS, +thiamine (P1171); lane 4: mcm7-GFPN760::Tcs strain containing pREP3X-TEV-NLS, -thiamine (P1171). All cultures were grown at 32°C. D. Fluorescence microscopy analysis of cells expressing Mcm7-GFPN760::Tcs in the absence (+thiamine) or presence (-thiamine) of TEV protease. Cells were fixed using methanol and acetone.
Yang et al. BMC Molecular Biology 2005 6:13 doi:10.1186/1471-2199-6-13