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.
Kobayashi
Teruaki
T
ZoeGene Corporation, Yokohama, Japan. kissou@max.hi-ho.ne.jp
Shiratori
Miwa
M
Nakano
Hirofumi
H
Eguchi
Chikashi
C
Shirai
Makoto
M
Naka
Daiji
D
Shibui
Tatsurou
T
D000818:Animals; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010455:Peptides / Q000378:metabolism*; D011691:Puromycin / Q000737:chemistry; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein
D010455:Peptides; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D011691:Puromycin; D005982:Glutathione Transferase