Construction of biotinylated firefly luciferases using biotin acceptor peptides

Analytical Biochemistry
H TatsumiY Koyama

Abstract

A cDNA for a thermostable mutant of Luciola lateralis (a Japanese firefly) luciferase was fused with either a gene for an artificial biotin acceptor peptide No. 84 [P. J. Schatz (1993) Bio/Technology 11, 1138-1143] or a gene for the carboxyl-terminal 87 residues of Escherichia coli biotin carboxyl carrier protein. The fused genes, when introduced into separate E. coli, directed the expression of luciferases that were able to bind with streptavidin. We purified them and showed that their specific activities and thermal stabilities remained unchanged. We also found that more than 95% of each fusion protein was biotinylated, suggesting that the biotin holoenzyme synthetase in the host cells worked efficiently. Using the biotinylated luciferases, we developed a highly sensitive bioluminescent enzyme immunoassay system.

Citations

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