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Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins

Waud, Jonathan P., Sala-Newby, G. B., Matthews, SB and Campbell, Anthony Keith 1996. Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1292 (1) , pp. 89-98. 10.1016/0167-4838(95)00199-9

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Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding fireflyluciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalentmodification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06–120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalentmodification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in the development of bioluminescent indicators of the covalentmodification of proteins.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: Q Science > QD Chemistry
Q Science > QR Microbiology
R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: Luciferase; Protein kinase; Bioluminescence; C-terminusengineering; Peptide; Proteinase
Publisher: Elsevier
ISSN: 0167-4838
Last Modified: 05 Jun 2017 02:58

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