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

Sala-Newby, G. B. and Campbell, Anthony Keith 1994. Stepwise removal of the C-terminal 12 amino acids of firefly luciferase results in graded loss of activity. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1206 (1) , pp. 155-160. 10.1016/0167-4838(94)90084-1

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Abstract

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Pharmacy
Subjects:	Q Science > QD Chemistry Q Science > QR Microbiology R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords:	Firefly luciferase; Bioluminescence; Protein engineering; C-terminus; Luciferase; (Firefly)
Publisher:	Elsevier
ISSN:	0167-4838
Last Modified:	24 Sep 2018 13:23
URI:	https://orca.cardiff.ac.uk/id/eprint/22489

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