Green fluorescent protein

Details Green fluorescent protein Green fluorescent protein

2000-02-29

2003-01-21

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007100, C435S004000, C530S350000, C530S300000, C436S517000

Reexamination Certificate

active

06509161

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method and apparatus for the detection of Green Fluorescence Protein GFP).
BACKGROUND TO THE INVENTION
GFP is found in the jellyfish
Aequorea victoria
. With the ability to clone and express GFP in a diverse range of cells and organisms including bacteria, yeast, plants and higher animals, GFP has become a versatile fluorescent marker for monitoring physiological processes, visualising protein localisation and detecting the expression of transferred genes [
Green Fluorescent Proteins, Proteins, Properties, Applications and Protocols
, ed. M. Chalfie and S. Kain, Wiley and Sons, 1
st
edn., 1998; H-H Gerdes and C. Kaether,
FEBS Lett.
, 1996, 389, 44; A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross and R. Y. Tsien,
Trends Biol. Sci.
, 1995, 20, 448]. The usefulness of GFP stems from the fact that fluorescence from GFP requires no additional co-factors; the fluorophore is self-assembling via a cyclization reaction of the peptide backbone.
GFP is bio-compatible, and when used as a tag does not alter the normal function or localisation of a protein to which it is fused. Proteins, cells and organelles marked with GFP can be visualised and monitored in living tissue without the need for fixation. Hence the dynamics of cellular processes can be non-invasively quantified in real time using GFP, simply by the measurement of fluorescence.
The wild-type GFP consists of 238 amino acids and has a cylindrical structure with the fluorophore element encapsulated in the centre [F. Yang, L. G. Moss and G. N. Phillips, Jr.,
Nat. Biotechol.
, 1996, 14, 1246]. As such it is a very chemically and photochemically stable and resilient fluorophore. Bright green fluorescence at 508-515 nm is readily induced by illumination of GFP with visible blue light at 470 nm. Genetic modification of GFP has been used to provide several useful mutants with fluorescence that is significantly blue or yellow shifted.
One application of GFP is in the development of an automated flow-injection bioassay for the detection of genotoxic compounds and the quantification of genotoxicity. The basis of the method is the use of yeast cells that are genetically modified such that they produce GFP in response to the activation of the cells' DNA repair mechanisms by DNA damage. The presence, concentration or potency, of a suspected genotoxic compound can be quantified by measuring an increase in green fluorescence from intact yeast cells [R. M. Walmsley, N. Billinton and W.-D. Heyer,
Yeast
, 1997, 13, 1535; N. Billinton, M. G. Barker, C. E. Michael, A. W. Knight, N. J. Goddard, P. R. Fielden and R. M. Walmsley,
Biosens. Bioelectron.
, 1998, 13, 831; A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton and R. M. Walmsley,
Meas. Sci. Technol.
, 1999, 10, 211].
Organs or organelles within organisms or cells where GFP is localised can often be readily visualised and distinguished from the background matrix by fluorescence microscopy techniques. However, in cases where GFP is only weakly expressed, or where GFP is in free solution such as in the cell cytosol, the fluorescence signal from GFP is invariably contaminated by cellular or media auto-fluorescence [K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson and D. W. Piston,
J. Microsc.
, 1995, 180, 109]. This has also been the case in yeast cell studies. In an analytical context this restricts the lower limit of detectable signal.
The term auto-fluorescence refers to fluorescence arising from any species other than GFP, known or unknown, naturally occurring or added, which is significantly bright at the wavelength of GFP fluorescence.
Green auto-fluorescence is almost universal to all living cells and organisms, and arises from a diverse range of sources. Likely chemical sources are reduced nicotinamide dinucleotides, oxidised flavins, age-related pigments and oxidised aromatic amino acids such as tryptophan. However, in many cases the exact source of auto-fluorescence is unknown. In general the brightness of the auto-fluorescence increases with the age of the cell or organism. Lipofuscins is a general term name for the auto-fluorescence that accumulates in (particularly ageing) mammalian cells. Auto-fluorescence has been noted to cause difficulties in the quantification of GFP expressed in many cells and species, for example:
Mammalian Heart Cells (Auto-fluorescence arising from myocardium); T. Kawada, W. S. Shin, Y. Nakatsuru, T. Koizumi, A. Sakamoto, T. Nakajima, Y. OkaiMatsuo, M. Nakazawa, H. Sato, T. Ishikawa, T. ToyoOka. Precise identification of gene products in hearts after in vivo gene transfection, using sendai virus-coated proteoliposomes.
Biochemical and Biophysical Research Communication
, 1999, 259, 408.
Plants (Auto-fluorescence arising from cell walls in maize plants); A. H. M. vanderGeest, J. F. Petolino. Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny.
Plant Cell Reports
, 1998, 17, 760.
Nematodes (Roundworms) (Auto-fluorescence mainly from the gut); S. Hashmi, M. A. AbuHatab, R. R. Gaugler. Green fluorescent protein a versatile gene marker for entomopathogenic nematodes,
Fundamental and Applied Nematology
, 1997, 20, 323
; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols
, ed. M. Chalfie and S. Kain, Wiley and Sons, 1
st
edn., 1998, p. 154.
Drosophila (Fruit flies); J. D. Plautz, R. N. Day, G. M. Dailey, S. B. Welsh, J. C. Hall, S. Halpain, S. A. Kay.
Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster
, plant and mammalian cells.
Gene
, 1996, 173, 83
; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols
, ed. M. Chalfie and S. Kain, Wiley and Sons, 1
st
edn., 1998, p. 172.
Bacteria; P. J. Lewis, J. Errington. Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in
Bacillus subtilis. Microbiology
-
UK
, 1996, 142, 733.
Yeast; Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1
st
edn., 1998, p. 149.
Many methods have been used to reduce the effect of auto-fluorescence upon GFP measurements, with varying degrees of success. However, each of the known methods suffers from disadvantages.
A first known method of reducing the effect of auto-fluorescence upon GFP measurements involves designing an optimised set of optical narrow-band filters to specifically pick out regions of the optical spectrum where GFP can be both excited, and emit light, with greater efficiency than the auto-fluorescence (see for example M. J. Zylka and B. J. Schnapp. Optimized filter set and viewing conditions for S65T mutant of GFP is living cells.
Biotechniques
, 1996, 21, 220). In most applications, the standard optical filter sets for fluorescein are not specific enough to discriminate GFP from auto-fluorescence. Dedicated excitation and emission filter sets for GFP have recently been made commercially available, although they are not suitable for all existing instrumentation. These filter sets suffer from the disadvantage that they are currently relatively expensive at several hundred pounds sterling per set. A further disadvantage is that in many cases the excitation and emission spectra of the auto-fluorescence significantly overlap those of GFP, making it difficult to distinguish between GFP fluorescence and auto-fluorescence signals.
Most researchers are required to optimise their own set of filters, using filters from various commercial sources, for their own particular applications. This is for two main reasons. Firstly, auto-fluorescence arises from a disparate, and often unknown range of chemicals, and varies enormously between different cells and species, and even over the lifetime of an individual cell. Secondly, new GFP mutants with diverse spectroscopic properties are becoming available all the time.
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