Fluorescent proteins

Details Fluorescent proteins Fluorescent proteins

2001-06-01

2004-11-16

Canella, Karen A. (Department: 1642)

Chemistry: molecular biology and microbiology

Vector, per se

C435S252300, C435S254110, C435S325000, C536S023100

Reexamination Certificate

active

06818443

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.
BACKGROUND OF THE INVENTION
The discovery that Green Fluorescent Protein (GFP) from the jellyfish
A. victoria
retains its fluorescent properties when expressed in heterologous cells has provided biological research with a new, unique and powerful tool (Chalfie et al (1994). Science 263:802; Prasher (1995) Trends in Genetics 11:320; WO 95/07463).
Furthermore, the discovery of a blue fluorescent variant of GFP (Heim et al. (1994). Proc.Natl.Acad.Sci. 91:12501) has greatly increased the potential applications of using fluorescent recombinant probes to monitor cellular events or functions, since the availability of probes having different excitation and emission spectra permits simultaneous monitoring of more than one process.
However, the blue fluorescing variant described by Heim et al, Y66H-GFP, suffers from certain limitations: The blue fluorescence is weak (emission maximum at 448 nm), thus making detection difficult, and necessitating prolonged excitation of cells expressing Y66H-GFP. Moreover, the prolonged period of excitation is damaging to cells especially because the excitation wavelength is in the UV range, 360 nm-390 nm.
A very important aspect of using recombinant, fluorescent proteins in studying cellular functions is the non-invasive nature of the assay. This allows detection of cellular events in intact, living cells. A limitation with current fluorescent proteins is, however, that relatively high intensity light sources are needed for visualization. Especially with the blue variant, Y66H-GFP, it is necessary to excite with intensities that are damaging to most cells. It is worth mentioning that some cellular events like oscillations in intracellular signalling systems, e.g. cytosolic free calcium, are very photo sensitive. A further consequence of the low light emittance is that only high levels of expression can be detected. Obtaining such high level expression may stress the transcriptional and/or translational machinery of the cells.
The excitation spectrum of the green fluorescent protein from Aequorea Victoria shows two peaks: A major peak at 396 nm, which is in the potentially cell damaging UV range, and a lesser peak at 475 nm, which is in an excitation range that is much less harmful to cells. Heim et al.(1995), Nature, Vol. 373, p. 663-4, discloses a Ser65Thr mutation of GFP (S65T) having longer wavelengths of excitation and emission, 490 nm and 510 nm, respectively, than the wild-type GFP and wherein the fluorophore formation proceeded about fourfold more rapidly than in the wild-type GFP.
Expression of GFP or its fluorescent variants in living cells provides a valuable tool for studying cellular events and it is well known that many cells, including mammalian cells, are incubated at approximately 37° C. in order to secure optimal and/or physiologically relevant growth. Cell lines originating from different organisms or tissues may have different relevant temperatures ranging from about 35° C. for fibroblasts to about 38° C.-39° C. for mouse &bgr;-cells. Experience has shown, however, that the fluorescent signal from cells expressing GFP is weak or absent when said cells are incubated at temperatures above room temperature, cf. Webb, C. D. et al., Journal of Bacteriology, October 1995, p. 5906-5911. Ogawa H. et al., Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 11899-11903, December 1995, and Lim et al. J. Biochem. 118, 13-17 (1995). The improved fluorescent variant S65T described by Heim et al. (1995) supra also displays very low fluorescence when incubated under normal culture conditions (37° C.), cf. Kaether and Gerdes FEBS Letters 369 (1995) pp. 267-271. Many experiments involving the study of cell metabolism are dependent on the possibility of incubating the cells at physiologically relevant temperatures, i.e. temperatures at about 37° C.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide novel fluorescent proteins, such as F64L-GFP (SEQ ID NO: 18, hereinafter referred to as F64L-GFP), F64L-Y66H-GPP (SEQ ID NO: 16, hereinafter referred to as F64L-Y66H-GFP) and F64L-S65T-GFP (SEQ ED NO: 20, hereinafter referred to as F64L-S65T-GFP) that result in a cellular fluorescence far exceeding the cellular fluorescence from cells expressing the parent proteins, i.e. GFP (SEQ ID NO: 22, hereinafter referred to as GFP), the blue variant Y66H-GFP and the S65T-GFP variant, respectively. This greatly improves the usefulness of fluorescent proteins in studying cellular functions in living cells.
A further purpose of the invention is to provide novel fluorescent proteins that exhibit high fluorescence in cells expressing them when said cells are incubated at a temperature of 30° C. or above, preferably at a temperature of from 32° C. to 39° C., more preferably at a temperature of from 35° C. to 38° C., and most preferably at a temperature of about 37° C.
It is known that fluorescence in wild-type GFP is due to the presence of a chromophore, which is generated by cyclisation and oxidation of the SYG at position 65-67 in the predicted primary amino acid sequence and presumably by the same reasoning of the SHG sequence and other GFP analogues at positions 65-67, cf. Heim et al. (1994). Surprisingly, we have found that a mutation, preferably a substitution, of the F amino acid residue at position 1 preceding the S of the SYG or SHG chromophore or the T of the THG chromophore, in casu position 64 in the predicted primary amino acid sequence, results in a substantial increase of fluorescence intensity apparently without shifting the excitation and emission wavelengths. This increase is remarkable for the blue variant Y66H-GFP, which hitherto has not been useful in biological systems because of its weak fluorescence.
The F64L, F64I, F64V, F64A, and F64G substitutions are preferred, the F64L substitution being most preferred, but other mutations, e.g. deletions, insertions, or posttranslational modifications immediately preceding the chromophore are also included in the invention, provided that they result in improved fluorescence properties of the various fluorescent proteins. It should be noted that extensive deletions may result in loss of the fluorescent properties of GFP. It has been shown, that only one residue can be sacrificed from the amino terminus and less than 10 or 15 from the carboxyl terminus before fluorescence is lost, cf. Cubitt et al. TIBS Vol. 20 (11), pp. 448-456, November 1995.
Accordingly, one aspect of the present invention relates to a fluorescent protein derived from Aequorea Green Fluorescent Protein (GFP) or any functional analogue thereof, wherein the amino acid in position 1 upstream from the chromophore has been mutated to provide an increase of fluorescence intensity when the fluorescent protein of the invention is expressed in cells. Surprisingly, said mutation also results in a significant increase of the intensity of the fluorescent signal from cells expressing the mutated GFP and incubated at 30° C. or above 30° C., preferably at about 37° C., compared to the prior art GFP variants.
There are several advantages of the proteins of the invention, including:
Excitation with low energy light sources. Due to the high degree of brightness of F64L-Y66H-GFP and F64L-GFP their emitted light can be detected even after excitation with low energy light sources. Thereby it is possible to study cellular phenomena, such as oscillations in intracellular signalling systems, that are sensitive to light induced damage. As the intensity of the emitted light from the novel blue and green emitting fluorescent proteins are of the same magnitude, it is possible to visualize them simultaneously using the same light source.
A real time reporter for gene expression in living cells is now possible, since the fluorescence from F64L-Y66H-GFP and F64L-GFP reaches a detectable level much faster than from wild type GFP, and prior known derivatives thereof. Hence, it is more suitable for real time studies of

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