tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.
We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.
Kurzchalia
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Zentralinstitut für Molekularbiologie der Akademie der Wissenschaften der DDR, Berlin-Buch.
Wiedmann
M
M
Breter
H
H
Zimmermann
W
W
Bauschke
E
E
Rapoport
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TA
D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000032:analysis*; D002474:Cell-Free System; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis*; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis*; D012357:RNA, Transfer, Lys; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D014908:Triticum
D000345:Affinity Labels; D011506:Proteins; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012357:RNA, Transfer, Lys; D001710:Biotin