Short peptide tags increase the yield of <Emphasis Type="Italic">C</Emphasis>-terminally labeled protein |
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Authors: | Teruaki Kobayashi Miwa Shiratori Hirofumi Nakano Chikashi Eguchi Makoto Shirai Daiji Naka Tatsurou Shibui |
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Institution: | (1) ZoeGene Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan;(2) Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, 3-21-1 Ami, Inashiki, Ibaraki 300-0393, Japan |
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Abstract: | 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 × Ala, 4 × His, 4 × Gln, and 4 × 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 × Ala and RGAA. Similar results were
obtained with various proteins and cell-free translation systems. |
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Keywords: | Cell-free translation Fluorescence Protein labeling Puromycin |
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