Cytosolic forms of beta-lactamase and uses thereof

Details Cytosolic forms of beta-lactamase and uses thereof Cytosolic forms of beta-lactamase and uses thereof

1996-10-15

2001-09-18

Low, Christopher S. F. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S018000, C435S325000, C435S320100, C536S023200

Reexamination Certificate

active

06291162

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to compositions and methods for use in measuring gene expression.
A reporter gene assay measures the activity of a gene's promoter. It takes advantage of molecular biology techniques, which allow one to put heterologous genes under the control of any promoter and introduce the construct into the genome of a mammalian cell [Gorman, C. M. et al.,
Mol. Cell Biol.
2: 1044-1051 (1982); Alam, J. and Cook, J. L.,
Anal. Biochem.
188: 245-254, (1990)]. Activation of the promoter induces the reporter gene as well as or instead of the endogenous gene. By design the reporter gene codes for a protein that can easily be detected and measured. Commonly it is an enzyme that converts a commercially available substrate into a product. This conversion is conveniently followed by either chromatography or direct optical measurement and allows for the quantification of the amount of enzyme produced.
Reporter genes are commercially available on a variety of plasmids for the study of gene regulation in a large variety of organisms [Alam and Cook, supra]. Promoters of interest can be inserted into multiple cloning sites provided for this purpose in front of the reporter gene on the plasmid [Rosenthal, N.,
Methods Enzymol.
152: 704-720 (1987); Shiau, A. and Smith, J. M.,
Gene
67: 295-299 (1988)]. Standard techniques are used to introduce these genes into a cell type or whole organism [e.g., as described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Expression of cloned genes in cultured mammalian cells. In:
Molecular Cloning
, edited by Nolan, C. New York: Cold Spring Harbor Laboratory Press, 1989]. Resistance markers provided on the plasmid can then be used to select for successfully transfected cells.
Ease of use and the large signal amplification make this technique increasingly popular in the study of gene regulation. Every step in the cascade DNA→RNA→Enzyme→Product→Signal amplifies the next one in the sequence. The further down in the cascade one measures, the more signal one obtains.
In an ideal reporter gene assay, the reporter gene under the control of the promoter of interest is transfected into cells, either transiently or stably. Receptor activation leads to a change in enzyme levels via transcriptional and translational events. The amount of enzyme present can be measured via its enzymatic action on a substrate. The substrate is a small uncharged molecule that, when added to the extracellular solution, can penetrate the plasma membrane to encounter the enzyme. A charged molecule can also be employed, but the charges need to be masked by groups that will be cleaved by endogenous cellular enzymes (e.g., esters cleaved by cytoplasmic esterases).
For a variety of reasons, the use of substrates which exhibit changes in their fluorescence spectra upon interaction with an enzyme are particularly desirable. In some assays, the fluorogenic substrate is converted to a fluorescent product. Alternatively, the fluorescent substrate changes fluorescence properties upon conversion at the reporter enzyme. The product should be very fluorescent to obtain maximal signal, and very polar, to stay trapped inside the cell.
To achieve the highest possible sensitivity in a reporter assay one has to maximize the amount of signal generated by a single reporter enzyme. An optimal enzyme will convert 10
5
substrate molecules per second under saturating conditions [Stryer, L. Introduction to enzymes. In:
Biochemistry
, New York: W. H. Freeman and company, 1981, pp. 103-134]. &bgr;-Lactamases will cleave about 10
3
molecules of their favorite substrates per second [Chang, Y. H. et al.,
Proc. Natl. Acad. Sci. USA
87: 2823-2827 (1990)]. Using a fluorogenic substrate one can obtain up to 10
6
photons per fluorescent product produced, depending on the type of dye used, when exciting with light of the appropriate wavelength. The signal terminates with the bleaching of the fluorophore [Tsien, R. Y. and Waggoner, A. S. Fluorophores for confocal microscopy: Photophysics and photochemistry. In:
Handbook of Biological Confocal Microscopy
, edited by Pawley, J. B. Plenum Publishing Corporation, 1990, pp. 169-178]. These numbers illustrate the theoretical magnitude of signal obtainable in this type of measurement. In practice a minute fraction of the photons generated will be detected, but this holds true for fluorescence, bioluminescence or chemiluminescence. A good fluorogenic substrate for a reporter enzyme has to have a high turnover at the enzyme in addition to good optical properties such as high extinction and high fluorescence quantum yield.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide &bgr;-lactamase substrate compounds. It is a further object of the invention to provide membrane-permeant compounds. The membrane-permeant compounds may be transformed into substantially membrane-impermeant compounds.
Another object of the invention is to provide &bgr;-lactamase reporter genes. A further object of the present invention is to create cells containing the &bgr;-lactamase reporter genes functionally linked to a promotor such that when the promotor is turned on, the reporter gene will be expressed. Expression of the &bgr;-lactamase is measured with the &bgr;-lactamase substrates which emit light after hydrolysis by the &bgr;-lactamase.
A further object of the invention is to use the &bgr;-lactamase reporter genes in cells and the &bgr;-lactamase substrate compounds of the present invention to screen for biochemical activity.
In accordance with the present invention, fluorogenic substrates are provided of the general formula I
wherein:
one of X and Y is a fluorescent donor moiety or a membrane-permeant derivative thereof, and the other is a quencher moiety, an acceptor fluorophore moiety or a membrane-permeant derivative thereof;
R′ is selected from the group consisting of H, lower alkyl, (CH
2
)
n
OH, (CH
2
)
n
COOR″, and ═NOJ, in which n is 0 or an integer from 1 to 5 and J is H, Me, CH
2
COOH, CHMeCOOH, and CMe
2
COOH;
R″ is selected from the group consisting of H, physiologically acceptable metal and ammonium cations, —CHR
2
OCO(CH
2
)
n
CH
3
, —CHR
2
OCOC(CH
3
)
3
, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, betamorpholinoethyl, dialkylaminoethyl, dialkylaminocarbonyloxymethyl, in which R
2
is selected from the group consisting of H and lower alkyl;
A is selected from the group consisting of S, O, SO, SO
2
and CH
2
;
Z′ is a linker for X; and
Z″ is a linker for Y.
In another aspect, this invention provides methods for determining whether a sample contains &bgr;-lactamase activity. The methods involve contacting the sample with a compound substrate of the invention, which exhibits fluorescence resonance energy transfer when the compound is excited; exciting the compound; and determining the degree of fluorescence resonance energy transfer in the sample. A degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of &bgr;-lactamase activity. One embodiment of this method is for determining the amount of an enzyme in a sample. According to this method, determining the degree of fluorescence resonance energy transfer in the sample comprises determining the degree at a first and second time after contacting the sample with the substrate, and determining the difference in the degree of fluorescence resonance energy transfer. The difference in the degree of fluorescence resonance energy transfer reflects the amount of enzyme in the sample.
In another aspect, this invention provides recombinant nucleic acid molecule comprising expression control sequences adapted for function in a vertebrate cell and operably linked to a nucleotide sequence coding for the expression of a &bgr

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