Biotin in vitro translation, nonradioactive detection of cell-free synthesized proteins.
In vitro translation of mRNAs into proteins is frequently used to study the coding capacity of RNAs or cDNAs and the functional effects of mutations. In vitro translation assays have traditionally been monitored by following the incorporation of a radiolabeled amino acid into newly synthesized protein. We have optimized an alternative nonradioactive biotin-labeling method. tRNALys is first aminoacylated with lysine, which is then chemically labeled with biotin. When biotin-lysine-tRNALys is added to translation systems, the biotinylated lysine is incorporated into the growing polypeptide chain. After electrophoresis and transfer to a blotting membrane, the biotin-labeled translation products are detected by a chemiluminescent reaction of luminol/iodophenol with streptavidin-coupled horseradish peroxidase. This nonradioactive method yields results equivalent to those obtained using the radioactive method. Biotin-labeled translation products are also biologically functional: (i) biotinylated precursor proteins are transported and processed correctly by dog pancreas microsomes; (ii) transcription factors synthesized by biotin in vitro translation bind specifically to their DNA recognition sequence; and (iii) biotin-modified luciferase keeps its enzymatic activity. The major advantage of the biotin in vitro translation system is that no radioactivity is required, and the method is easy, economical, reproducible and fast--the whole nonradioactive procedure, from translation to detection, can be completed within six hours.
Hoeltke
H J
HJ
Boehringer Mannheim GmbH, Penzberg, Germany.
Ettl
I
I
Strobel
E
E
Leying
H
H
Zimmermann
M
M
Zimmermann
R
R
D000818:Animals; D001710:Biotin; D002474:Cell-Free System; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D010179:Pancreas / Q000737:chemistry; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D010455:Peptides; D012333:RNA, Messenger; D012357:RNA, Transfer, Lys; D014157:Transcription Factors; D001710:Biotin; D008156:Luciferases; C013411:biocytin; D008239:Lysine