BIOTIN IN-VITRO TRANSLATION, NONRADIOACTIVE DETECTION OF CELL-FREE SYNTHESIZED PROTEINS

In vitro translation of mRNAs into proteins is frequently used to stud y the coding capacity of RNAs or cDNAs and the functional effects of m utations. In vitro translation assays have traditionally been monitore d by following the incorporation of a radiolabeled amino acid into new ly synthesized protein. We have optimized an alternative nonradioactiv e biotin-labeling method. rRNA(Lys) is first aminoacylated with lysine , which is then chemically labeled with biotin. When biotin-lysine-tRN A(Lys) is added to translation systems, the biotinylated lysine is inc orporated into the growing polypeptide chain. After electrophoresis an d transfer to a blotting membrane, the biotin-labeled translation prod ucts 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 radioact ive method. Biotin-labeled translation products are also biologically functional: (i) biotinylated precursor proteins are transported and pr ocessed correctly by dog pancreas microsomes; (ii) transcription facto rs synthesized by biotin in vitro translation bind specifically to the ir DNA recognition sequence; and (iii) biotin-modified luciferase keep s its enzymatic activity. The major advantage of the biotin in vitro t ranslation system is that no radioactivity is required and the method is easy, economical, reproducible and fast-the whole nonradioactive pr ocedure, from translation to detection, can be completed within six ho urs.