Construction of Biotinylated Firefly Luciferases Using Biotin Acceptor Peptides

✍ Scribed by Hiroki Tatsumi; Satoshi Fukuda; Mamoru Kikuchi; Yasuji Koyama

• Book ID: 102563453
• Publisher: Elsevier Science
• Year: 1996
• Tongue: English
• Weight: 119 KB
• Volume: 243
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.0498

✦ Synopsis

little as 100 ng/ml of human immunoglobulin G (IgG) A cDNA for a thermostable mutant of Luciola laterwith EIA (6).

alis (a Japanese firefly) luciferase was fused with ei-

Recently a method to biotinylate proteins by means ther a gene for an artificial biotin acceptor peptide No. of genetic engineering became available. Biotin en-84 [P. J. Schatz (1993) Bio/Technology 11, 1138-1143] zymes such as acetyl-CoA carboxylase and pyruvate or a gene for the carboxyl-terminal 87 residues of Eschcarboxylase contain a biotin molecule which is covaerichia coli biotin carboxyl carrier protein. The fused lently attached to a unique Lys via an amino linkage genes, when introduced into separate E. coli, directed catalyzed by biotin holoenzyme synthetase (7). A highly the expression of luciferases that were able to bind conserved sequence was observed around the biotinwith streptavidin. We purified them and showed that attachment site. Proteins which were genetically fused their specific activities and thermal stabilities rewith the biotin acceptor peptides were biotinylated at mained unchanged. We also found that more than 95% the Lys in vivo (8-11).

of each fusion protein was biotinylated, suggesting

Another problem about PpL is thermal stability. It that the biotin holoenzyme synthetase in the host cells retains less than 50% of the initial activity after 30 worked efficiently. Using the biotinylated luciferases, min at 40ЊC (12). This instability makes it difficult to we developed a highly sensitive bioluminescent enuse PpL for EIA. Recently Kajiyama and Nakano obzyme immunoassay system.