Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1997-10-24
2002-12-10
Arthur, Lisa B. (Department: 1634)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S008000, C435S029000
Reexamination Certificate
active
06492500
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to bioluminescent proteins, in particular it relates to bioluminescent proteins which have been modified, for example by chemical means or by genetic engineering. Such modified bioluminescent proteins, hereinafter referred to as “rainbow proteins”, may be used in the detection and quantification of cells, microbes such as bacteria, viruses and protoza, and substances of biological interest such as substrates, metabolites, intra- and extra- cellular signals, enzymes, antigens, antibodies and nucleic acids.
Bioluminescence is the oxidation of an organic molecule, the “luciferin”, by oxygen or one of its metabolites, to emit light. The reaction is catalysed by a protein, usually known as a “luciferase”, or a “photoprotein” when the luciferin is so tightly or covalently bound to the luciferase that it does not diffuse off into the surrounding fluid. O
2
+lucifern+luciferase→oxylucifern+light (or O
−
2
or H
2
O
2
) (or photoprotein)
Hp to three other substances may also be required to be present in order to generate light, or to alter its colour, and they are as follows:
(a) A cation such as H
+
, Ca
2+
, Mg
2+
, or a transition metal such as Cu
+
/Cu
2+
, Fe
2+
/Fe
3+
.
(b) A cofactor such as NADH, FMN, or ATP.
(c) A fluor as an energy transfer acceptor.
Five chemical families of luciferin have been identified so far (see
FIG. 1
of the attached drawing):
(a) Aldehydes (found in bacteria, freshwater limpet Latia and earthworms).
(b) Imidazolopyrazines (found in Sarcomstigophora, Onidaria, Ctenophora, some Arthorpoda, some Mollusca, some Chordata).
(c) Benzothiazole (found in beetles such as fireflies and glowworms).
(d) Linear tetrapyrroles (found in dinoflagellates, euphasiid shrimp, some fish).
(e) Flavins (found in bacteria, fungi, polychaete worms and some molluscs).
Reactions involving these luciferns may result in the emission of violet, blue, blue-green, green, yellow or red light and occasionally UV or IR light and such emission may or may not be linearly or circularly polarised. Reference is directed to Chemiluminescence principles and applications in biology and medicine, A. K. Campbell, publ. 1988 Horwood/VCH Chichester Weinheim, for further discussion of bioluminescent reactions.
It has now been found that the light emitted from a bioluminescent reaction involving a modified bioluminescent or “rainbow” protein, may be changed in intensity, colour or polarisation. Such a change can then be used in various assays for detecting, locating and measuring cells, microbes and biological molecules.
In this instance, the cell or substance causes a physical or chemical change, such as phosphorylation, to a rainbow protein such as a genetically engineered luciferase, resulting in a change in intensity, colour or polarisation of the light emission. The bioluminescent reaction is triggered by adding, for example, the luciferin, and modification of the luciferan by the cell or substance being measured causes the reaction to emit light at a shorter or longer wavelength. This enables specific reactions to be detected and quantified in live cells, and within organelles or on the inner or outer surface of the plasma membrane, without the need to break them open, and without the need for separation of bound and unbound fractions.
SUMMARY OF THE INVENTION
According to one aspect of the invention there is provided a bioluminescent protein capable of taking part in a bioluminescent reaction to produce light or radiation of altered characteristics under different physical, chemical, biochemical or biological conditions.
The rainbow protein may be produced by the alteration, substitution, addition or removal of one or more amino acids from the end of or within the luciferase or photoprotein. As a result the light emission from the oxyluciferin may be of different colours or different states of polarisation depending on the physical or chemical conditions. A change in colour to another part of the rainbow spectrum may be induced by:
(a) A change in cation concentration such as 4, Ca Mg, or transition metal.
(b) A change in anion concentration such as C1
−
or phosphate.
(c) Covalent modification of the new protein by enzymes causing phospho- or dephosphorylation (including ser/thr, his, and tyr kinases and phosphatases) transglutamination, proteolysis, ADP ribosylation, gly- or glu-cosylation, halogenation, oxidation, methylation and myristilation.
(d) Binding to the rainbow protein of an antigen, an intracellular signal such as cyclic AMP, cyclic GMP, Ip3, Ip4, diacyl glycerol, ATP, ADP, AMP, GTP, any oxy or deoxyribonucloside or nucleotide, a substrate, a drug, a nucleic acid, a gene regulator protein.
(e) Expression of its nucleic acid inside a live cell, as well as its modification or regulation within cell by gene expression such as promoters, enhancers or oncogenes.
Single or multiple mutations and deletions may be detected in a piece of DNA (eg a PCR product) by linking the “rainbow protein” to one end of the DNA and an energy transfer acceptor or quencher to the other end. Nuclease attack at the mutation will separate the rainbow protein from the acceptor or quencher and thus cause a change in intensity, colour or polarisation of the light emission.
Such alteration, substitution, addition or removal of one or more amino acids may be achieved by chemical means. Alteration of an acid includes alkylation (eg. methylation), phosphorylation and various other covalent modifications of the type outlined herein. Alternatively the nucleic acid coding for the luciferase or photoprotein may be altered by modifying, substituting, inserting or deleting one or more nucleotides such that the resulting protein has gained or lost a site which interacts with the cations, anions, intracellular signal, covalent modification; proteins or nucleic acid to be measured. The insertion or deletion of nucleotides is normally produced by site directed mutagenesis or by opening up the gene with a specific restriction enzyme, inserting or deleting a selected nucleotide sequence and then sealing up of the gene again or using specific primers with the polymerase chain reaction. The nucleic acid is then transcribed to mRNA and this is then translated to form the rainbow protein either inside a bacterial or eukaryotic cell, or in vitro using, for example, rabbit reticulocyte lysate. The new nucleic acid may contain an RNA polymerase promoter such as T7, SP6, or mammalian promotors such as actin, myosin, myelin proteins, MMT-V, SV40, antibody, G6P dehydrogenase, and can be amplified in vitro using the polymerase chain reaction. The result is that the rainbow protein can be produced either in a live cell such as a cancer cell, or without cells using enzymatic reactions in vitro. The addition of tissue specific promoter or enhancer sequences to the 5′ or 3′ end of the DNA coding for the native or altered bioluminescent protein will enable it to be used as a reporter gene and to be expressed specifically in a particular cell or tissue, the expression being detectable by the appearance of a change in light intensity, colour or polarisation.
Another way of producing the DNA for a rainbow protein is to separate into two halves the original DNA for the bioluminescent protein. A piece of DNA or gene is then inserted between the two halves by ligating one half of the 5′ end and the other to the 3′ end. Alternatively the rainbow protein DNA could be generated using the polymerase chain reaction so that the sense primer had one part of the rainbow protein DNA linked at 5′ end and the antisense primer and the other part linked at the 3′ end (i.e. antisense). The pieces of DNA or gene of interest, in the middle, could be from two separate genes. For example one could code for an energy transfer protein, the other for a bioluminescent protein. Only when the two are linked together via a peptide (from DNA/RNA) will the rainbow protein be generated and a shift in colour occur. The ene
Arthur Lisa B.
University of Wales College of Medicine
Young & Thompson
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