Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions.
To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.
Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan. Electronic address: sinouye@jnc-corp.co.jp.
Satoshi
S
Inouye
Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan.
Jun-Ichi
J
Sato
Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan.
Yuiko
Y
Sahara-Miura
Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Suguru
S
Yoshida
Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Electronic address: thosoya.cb@tmd.ac.jp.
Takamitsu
T
Hosoya
D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D033364:Decapoda / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D015394:Molecular Structure; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D007093:Imidazoles; D050505:Mutant Proteins; D011719:Pyrazines; D011994:Recombinant Proteins; C017144:coelenterazine; D008156:Luciferases
Coelenterazine analogues; Inclusion bodies; Mutagenesis; Secretion