Skip to main content
. 2012 Apr 13;7(4):e33042. doi: 10.1371/journal.pone.0033042

Figure 4. Expression of ALA1 in yeast requires optimization of the cDNA sequence.

Figure 4

(A) Total membrane protein preparations (30 µg) were run on SDS-PAGE gels and subjected to immunodetection with antibodies raised against the engineered epitope tags. Only ALIS proteins and yeALA1 can be detected with the anti-RGSH antibody. (B) ALA1 cDNA was modified to eliminate a putative transcription termination site and five rare codons codifying for Arg (yeALA1) and expressed alone or together with an ALIS in a yeast Δdrs2Δdnf1Δdnf2 deletion mutant. ALA1 was tagged at the N-terminal end with an RGSH10-tag while ALIS proteins bear a RGSH6-tag at the same end. ALA1 and ALA1noTT (ALA1 cDNA codified to eliminate a putative transcription termination site) were also expressed under the same conditions. Total membrane protein preparations (30 µg) were run on SDS-PAGE gels and subjected to immunodetection with antibodies raised against the engineered epitope tags. Bands corresponding to ALIS proteins are immunodecorated with an anti-RGSH antibody, while an anti-HA antibody cannot recognize any band corresponding to HA-tagged ALA1.