Stepwise removal of the C-terminal 12 amino acids of firefly luciferase results in graded loss of activity.

The C-terminus of the firefly luciferase (550 amino acids) was engineered using PCR followed by in vitro transcription-translation in order to investigate the role of the last 12 amino acids in the bioluminescence. Coding sequences were removed stepwise and the decapeptide MRSAMSGLHL, a putative AMP-activated protein kinase phosphorylation site, was used to replace the last 8-12 amino acids in order to test for amino acid specificity at the C-terminus. Removal of up to seven of the C-terminal amino acids resulted in no detectable loss of bioluminescent activity. However, the luciferase activity decreased stepwise from 50 to 0.1% when 8-12 amino acids were removed. Replacement of amino acids 539-550 and 543-550 by MRSAMSGLHL generated luciferases that retained 22 and 35% of catalytic activity respectively. These results have important implications for the further development of engineered luciferases as intracellular indicators and the understanding of the active centre of beetle luciferases.