Short peptide tags increase the yield of C-terminally labeled protein.

C-Terminal protein labeling allows non-radioactive detection of proteins by using fluorescent puromycin derivatives and cell-free translation systems. However, yields of some labeled proteins are low. Here, we report that the yield of labeled protein mainly depends on the C-terminal amino acid sequence. The short peptide tag sequence, RGAA, at the C-terminus increased not only the labeling efficiency (more than 80%) but also the synthesis yield of labeled proteins. To examine the relationship between the C-terminal amino acid sequence and the yield of labeled proteins, we synthesized C-terminally labeled glutathione S-transferase (GST) containing four identical amino acid residues at the C-terminus. The results demonstrated that 4 x Ala, 4 x His, 4 x Gln, and 4 x Cys produced over 200% of the yield of wild-type GST. In addition, the two Ala residues produced almost the same synthesis activity as 4 x Ala and RGAA. Similar results were obtained with various proteins and cell-free translation systems.