Folding of firefly luciferase during translation in a cell‐free system

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell‐free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell‐free system while this protein is being translated from its mRNA. It is shown that ribosome‐bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half‐time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.