Oplophorus luciferase subunits

Details Oplophorus luciferase subunits Oplophorus luciferase subunits

2001-04-26

2003-04-08

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving luciferase

C435S189000, C530S350000

Reexamination Certificate

active

06544754

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel luciferase derived from Decapoda. More specifically, the invention relates to a secretional luciferase from
Oplophorus gracilirostris
, which is composed of 19 kDa and 35 kDa proteins. The invention also relates to polynucleotides encoding at least one of these proteins, a recombinant expression vector comprising at least one of these polynucleotides, a host cell transformed with said vector, and process for producing said photoprotein.
BACKGROUND OF THE INVENTION
The genes encoding luciferases or photoproteins which have been reported are listed in Table 1.
TABLE 1
Reported genes of photoproteins and luciferases
Discoverer
(publication
Protein
Origin
year)
Gene Accession No.
1. Photoprotein
Aequorin
Aequorea victoria
Inouye et al.
AEVAQ440X: L29571
(1985)
Aequorea victoria
Prasher et al.
AEVAEQA: M16103
(1987)
Clytin
Clytia gregarium
Inouye & Tsuji
CY1APOCLYT:
(1993)
L13247
Mitrocomin
Mitrocoma
Fagan et al.
MITMI17: L31623
cellularia
(1993)
Obelin
Obelia longissima
Illarionov et al.
OLU07128: U07128
(1995)
2. Luciferase
Firefly
Photinus pyralis
de Wet et al.
PPYLUC: M15077
(1987)
Luciola cruciata
Matuda et al.
FFLLUC: M26194
(1989)
Luciola lateralis
Tatsumi et al.
LLUCI: X66919
(1992)
Luciola lateralis
Cho et al.
LLLUCIFMJ: Z49891
(1995)
Luciola
Devine et al.
mingrelica
(1993)
Photuris
Zenno et al.
D25415: D25415
pennsylvanica
(1993)
Photuris
Ye et al. (1997)
PPU31240: U31240
pennsylvanica
Pyrocoelia
Ohmiya et al.
PIBLUCA: L39928
miyako
(1995)
Hotaria parvula
Ohmiya et al.
HOTLUCI: L39929
(1995)
Glow worm
Lampyris
Sala-Newby et
LNLUCPROT:
noctiluca
al. (1996)
X89479
Click beetle
Pyrophorus
Wood et al.
plagiophthalamus
(1989)
Railroad-
Phrixothrix
Viviani et al.
AF139644: AF139644
worm
vivianii
(1999)
Phrixothrix hirtus
Viviani et al.
AF139645: AF139645
(1999)
Vargula
Vargula
Kazami et al.
Pat. Appln. No. JP63-
hilgendorfii
(1988)
199295
Thompson et
VAHLUC: M25666
al. (1989)
Renilla
Renilla reniformis
Lorenz et al.
RELLUC: M63501
(1991)
Gonyaulax
Gonyaulax
Bae & Hastings
GONLUCA: L04648
polyedra
(1994)
Bacteria
Vibrio fischeri
Foran & Brown
VFLUXAB: X06758
(1988)
Vibrio harveyi
Cohn et al.
VIBHALUXA:
(1985)
M10961
Johnston et al.
VIBHALUXA:
(1986)
M10961
Photobacterium
Illarionov et al.
PLLUXABG: X08036
leiogathis
(1988)
Lee et al.
PHRLUX: M63594
(1991)
Photobacterium
Ferri et al.
PHRLUXABDF:
phosphoreum
(1991)
M65067
Xenorhabdus
Johnston et al.
XENLUXABB:
luminescence
(1990)
M55977
Szittner &
XENLUXAB:
Meighen
M57416
(1990)
Kryptophanaron
Haygood
KRYLUC: M36597
alfredi
(1986, 1990)
Alteromonas
Zenno et al.
Pat. Appln. No. JP06-
hanedai
(1994)
035450
These photoproteins and luciferases are an industrially important protein and have been utilized, for example, as a reporter enzyme. Various methods for detecting an analyte using luminescent reactions of these enzymes have been developed, and also some apparatuses to be used in these methods have been improved and widespread. Among these known photoproteins and luciferases, however, there is no enzyme applicable to extensive purposes. Consequently, one has to choose a proper enzyme for individual purpose.
Among the prior art luminescent substrates (often referred to as luciferin), those having the determined structures are only the substrates represented by formulas (1)-(8):
The luminescent substrates include species specific and species non-specific substrates. The minimum unit in the enzymatic bioluminescent reaction consists of a luminescent enzyme (luciferase), a luminescent substrate (luciferin) and molecular oxygen. A luminescent reaction which requires other components such as a co-enzyme or a supplemental molecule is also reported.
Examples of the luciferase with luminescence in minimum unit include those derived from Renilla, Cypridina and Gonyaulax. The luciferins corresponding to these luciferases have very complicated structures as shown in the above formulas (4) and (5). The methods for synthesizing Cypridina and Gonyaulax luciferins are already known, but yield is remarkably low due to their complicated synthesizing process. Though the luciferins extracted from natural products are,also used, they are very expensive with little industrial utility.
On the contrary, Renilla luciferin known as coelenterazine and derivatives thereof are commercially available and inexpensive, because various methods for the production thereof have been established.
Among the photoproteins in Table 1, a secretional luciferase is only Cypridina luciferase. The structure of the gene is reported in Thompson, E. M., et al., Proc. Natl. Acad. Sci. USA, 86, 6567-6571 (1989) and the application of the gene is reported in Inouye, S., et al., Proc. Natl. Acad. Sci. USA, 89, 9584-9587 (1992).
In the construction of a bioassay system such as a drug screening system, the secretional luciferase has an industrial advantage in that the luminescence activity can be detected in living cells without cell disruption using the luciferase extracellularly secreted as a reporter. Generally, a secretional protein is not particularly difficult to produce if a suitable host-vector system is selected. Further, the purification of a recombinant protein from cultured media is easier in comparison with the purification from a cell extract. Thus, mass production of the secretional luciferase may advantageously hold down purification costs involved.
A particularly useful luciferase involves a luminescent system wherein the luminescent reaction occurs only among the minimal unit, i.e. a luciferase, a luciferin and molecular oxygen, the luciferin being coelenterazine or a derivative thereof which is readily available, and the luciferase itself being a secretional protein. A protein and a gene of such luciferase are advantageous not only scientifically but also industrially. However, isolation of gene encoding such luciferase and expression thereof in a living cell has not been reported yet.
A luciferase derived from a luminous shrimp belonging to Decapoda has been reported as a secretional luciferase, the luminescent substrate of which is coelenterazine. It is known that a luminous shrimp involves a secretional luciferase (enzyme) and that a blue luminescence is emitted by the reaction of the luciferase, a luminescent substrate luciferin and molecular oxygen.
The detailed classification of globally living luminous shrimps is disclosed in Herring, P. J., J. Mar. Biol. Ass. U.K., 56, 1029-1047 (1976). The only biochemical study of luciferase of luminous shrimp is reported by Shimomura et al., on a luciferase of the luminous shrimp
Oplophorus gracilirostris
living in the Suruga Bay, Sizuoka, Japan (Biochemistry, 17, 994-998 (1978)). This study report discloses a luciferase having a molecular weight of 130,000 which is composed of the tetramer of a polypeptide having a molecular weight of 31,000. The literature also reports that the luciferase has a quantum yield of 0.32 at 22° C., a high specific activity of 1.75×10
15
Photons/s. mg, an optimum light emission at 40° C. and an excellent heat stability. It also describes that the luminescent reaction proceeds in a wide range of pH.
The luciferin in the luminescent reaction of Oplophorus luciferase is coelenterazine represented by the above formula (2), which is also a luminescent substrate in the luminescent reactions of Renilla luciferase and a photoprotein, aequorin. The most important difference between these luminescent enzymes and Oplophorus luciferase is that Oplophorus luciferase has very broad substrate specificity in comparison with those of Renilla luciferase and aequorin. Oplophorus luciferase is more preferable than other luciferases, because it can utilize as a substrate bisdeoxycoelenterazine which is an analogue of coelenterazine and is available at a low cost.
However, either the protein structure or the gene structure of Oplophorus luciferase of a secretional type has not been elucidated. This is because living luminous shrimp, which are mostly living in the deep-sea, are very difficult to obtain in a large amount. Furthermore, popula

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