Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
1999-08-25
2001-07-24
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S189000, C435S252300, C435S320100, C435S440000, C435S810000, C536S023200
Reexamination Certificate
active
06265177
ABSTRACT:
TECHNICAL FIELD
The present invention relates broadly to enzymes and methods suitable for assaying ATP. It further relates to specific applications for such assays.
BACKGROUND ART
Intracellular ATP concentrations can vary 10-fold or more depending upon a cell's state of health or developmental stage. It is of great value to be able to measure fluctuations in intracellular ATP levels as a means of investigating e.g. the effects of drugs, toxins, hormones, environmental agents or disease on cells.
There is apparently at present no convenient method for analysing the concentration of ATP in vivo. For instance, in Dementieva et al (1996) Biochemistry (Moscow) Vol 61, No. 7., the intracellular concentration of ATP was measured in
E. coli
by calculating the total amount of ATP present using a recombinant luciferase, and dividing by an estimated total cell volume.
Such an indirect approach can at best produce only an estimate of the actual ATP concentration.
The measurement of ATP concentration in cells has also been performed using an in vitro coupled assay, such as that disclosed in the Sigma Diagnostic Kit Catalog No. 366, in which Phosphoglycerate kinase is used to convert 3-phosphoglycerate to 1,3 diphosphoglycerate in an [ATP]-dependent fashion. The 1,3 diphosphoglycerate is then converted to glyceraldehyde-3-P concommitantly with conversion of NADH to NAD, which can be monitored spectroscopically. The assay has a dynamic range up to 1 mM; the expected range is 380-620 &mgr;m when used with blood cells.
However it can be seen that, as with all coupled assays, the test is inevitably cumbersome to perform. Additionally it could not readily be adapted for in vivo use. It would thus be a contribution to the art to provide materials and methods which overcome some of the drawbacks of the prior art.
DISCLOSURE OF THE INVENTION
In a first aspect of the invention there is provided a recombinant mutant luciferase having a mutation which is such that the K
m
for ATP of the luciferase is increased with respect to that of the corresponding non-mutated enzyme. Preferably the K
m
is at least double that of the non-mutated enzyme, and more preferably at least around five, ten, or twenty times higher than that of the non-mutated enzyme.
Luciferases are, of course, already known in the art. In the presence of Mg
2+
, luciferase (originally obtained from fireflies) catalyzes the reaction of luciferin, ATP and O
2
to form oxyluciferin, AMP, CO
2
, pyrophosphate and light. This basic property (luciferin and ATP to produce light) is hereinafter referred to as ‘luciferase activity’.
The term ‘luciferase’ as used in relation to the invention is intended to embrace all luciferases, or recombinant enzymes derived from luciferases which have luciferase activity. This explicitly includes recombinant mutant luciferases which have deletions, additions or substitutions to their amino acid structure provided that they retain luciferase activity. Such luciferases will typically have considerable homology (e.g. up to 70, 80, 90, or 99%) with wild-type enzymes. However the crucial technical feature of the luciferases of the present invention which distinguishes them from those of the prior art is that they have a mutation which causes an increase in the K
m
for ATP of the luciferase as compared with that measured for a corresponding enzyme which differs only in it that it lacks that same mutation.
This increase K
m
may be measured by the person of ordinary skill in the art by conventional enzyme assays, as described in more detail in the Examples below.
It should be noted that in the prior art, luciferase has sometimes been used as a marker for gene expression (in vivo) where its production in a cell is linked to a particular genetic control element. Luciferin is added exogenously and intracellular ATP concentrations, under almost all conditions, will be such that the enzyme is saturated. Thus the switching on of gene expression is signalled by light that is emitted in a quantitative manner according to the amount of active luciferase that is generated.
However it should be stressed that in the previously known systems it is generally the concentration of luciferase which is measured; this concentration is then correlated with a different event e.g. the efficiency of a promoter. Indeed it has, on occasions, been an object of the prior art teaching on luciferases to reduce the K
m
for ATP see e.g. WO 96/22376) which ensures that changes in the ambient [ATP] does not interfere with the assay.
Similarly the assay disclosed by Dementieva et al (1996) discussed above requires that all of the ATP be efficiently converted to light so that the total ATP present can be calculated. This approach requires a low K
m
luciferase so that the enzyme operates at near maximal velocity until all the ATP is hydrolysed.
By making available luciferases which have an increased K
m
compared with those already known in the art, the present inventors have for the first time opened up the possibility of using these enzymes to measure steady state ATP concentrations over range which was previously unsuitable. This is because, generally speaking, the relationship between enzyme velocity (V, as measured by light intensity) and substrate concentration (of ATP, where luciferin is in excess) is as follows:
V=V
m.
[ATP]/
K
m
+[ATP]
It can therefore be seen that only when the K
m
is greater than (or of a similar order as) the ambient [ATP] will there be a degree of proportionality between changes in [ATP] and changes in light intensity. Where the K
m
is much less than the ambient [ATP], any changes in [ATP] will not tangibly effect the measured light intensity. Clearly the more sensitive the light detection is, the smaller the measurable changes in ‘V’ can be, and the smaller the K
m
can be with respect to the [ATP] range being assessed.
For certain applications, e.g. in vivo measurements, it may be advantageous to have a luciferase wherein the K
m
is of the order of between 400 &mgr;m to 1.4 mM e.g. 500 &mgr;m, 600 &mgr;m, 1 mM etc. However, as can be appreciated from the discussion above, the main criterion is that the K
m
is not much less than the expected [ATP] range to be assessed, and the phrase ‘of the order of’ should be construed accordingly.
A particular expected [ATP] range which is important for physiological assays of blood cells is between 300 &mgr;m and 1 mM, or more particularly 380 &mgr;m and 620 &mgr;m, (cf. Sigma Diagnostic Kit, Catalog No. 366 discussed above). For other mammalian cells such as hepatocytes, the [ATP] range is 2.5 mM-6 mM (see Dementieva et al (1996) discussed above. Use of the recombinant luciferases of the present invention for continuous assays in these ranges is particularly envisaged.
The disclosure of the present application makes such high K
m
luciferases available for the first time. The prior art disclosures reveal only luciferases having a K
m
of between 60 &mgr;m and 150 &mgr;m, which would be saturated in these ranges.
It is also advantageous, as with all enzymes used in assays, that the mutant enzyme retains sufficient activity (i.e. a high maximum turnover number, giving a high V
m
) such that practical concentrations of enzyme can give detectable results.
Preferably the activity for ATP of the mutant is at least 5-100% of that of the corresponding wild-type; however reduced-activity as a result of the high K
m
mutation can, if necessary, be compensated for by using more enzyme or more sensitive detection if required.
In one embodiment of the first aspect there is disclosed a luciferase wherein the amino-acid corresponding to amino acid residue number 245 in
Photinus pyralis
has been substituted with respect to the corresponding wild-type amino acid residue such that the K
m
for ATP is increased with respect to that of the corresponding non-mutated enzyme.
It should be noted that the sequences of a number of luciferases from dif
Lowe Christopher Robin
Murray James Augustus Henry
Squirrell David James
White Peter John
Nixon & Vanderhye P.C.
Slobodyansky Elizabeth
The United States of America as represented by the Secretary of
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