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.
Sala-Newby
G B
GB
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Campbell
A K
AK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D001517:Coleoptera / Q000201:enzymology*; D008156:Luciferases / Q000737:chemistry*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry
D000596:Amino Acids; D010455:Peptides; D011994:Recombinant Proteins; D008156:Luciferases