Biotin in vitro Translation: A Nonradioactive Method for the Synthesis of Biotin Labeled Proteins in a Cell-Free System

Summary

In vitro translation of mRNAs into proteins using, for example, reticulocyte lysates or wheat germ extracts is frequently applied to study the coding capacity of RNAs or cDNAs and the functional effects of mutations. Usually, a natural and purified RNA or an mRNA transcript synthesized from cloned cDNA in vitro with a phage RNA polymerase (SP6, T3, or 17) is added to an in vitro translation system in order to program the synthesis of the encoded protein. In vitro translation assays are traditionally monitored by following the incorporation of radiolabeled [³⁵S]methionine into newly synthesized protein. We have optimized an alternative nonradioactive in vitro translation method that labels newly synthesized proteins with biotin. tRNA^Lys is first aminoacylated with lysine. The lysine moiety is then chemically labeled with biotin at the E NH₂-group. When biotin-lysine-tRNA^Lys is added to translation systems, the biotinylated lysine is incorporated into the growing polypeptide chain. After electrophoresis and transfer of the translation products to PVDF or nitrocellulose membranes, the biotin-labeled proteins are detected with streptavidin coupled to horseradish peroxidase and a chemiluminescent reaction of the marker enzyme with luminol/iodophenol. The chemiluminescent signals are recorded by a 0.1 to 10-minute exposure of the blot to X-ray film. For the translation of viral and in vitro transcribed RNAs, this nonradioactive method yields equivalent results in comparison to the radioactive method. In addition, biotin-labeled translation products are biologically functional: biotinylated precursor proteins are transported and processed correctly by dog pancreas microsomes; transcription factors synthesized by biotin in vitro translation bind specifically to their DNA recognition sequences; and 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 6 hours.