Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-05
2001-12-04
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S008000, C435S015000, C435S007500
Reexamination Certificate
active
06326157
ABSTRACT:
BACKGROUND OF THE INVENTION
The
Aequorea victoria
green fluorescent protein's (GFP) rising popularity in recent years can be attributed to its utility as both a nontoxic marker for gene expression and as a fluorescent fusion protein tag, primarily for use with fluorescence microscopy (FM) or flow cytometry (FC). To date, these methods have been used to provide qualitative information on a tagged gene product's expression level and/or cell locale.
With the growing use of GFP for studies of gene expression, and with the development of brighter, wavelength-shifted mutant GFPs which have enabled the tailoring of its standard use to conventional flow cytometry and fluorescence microscopy lasers and/or filter sets, it has become important to develop procedures for quantitative GFP analysis in flow cytometers. See, e.g., “Use of the Green Fluorescent Protein and its Mutants in Quantitative Fluorescence Microscopy,” by George H. Patterson et al., Biophysical Journal 73, 2782 (1997), which discusses the spectroscopy of GFP in bulk samples, and “Green Fluorescent Protein as a Marker for Gene Expression,” by Martin Chalfie et al., Science 263, 802 (1994). The difficulty of attaching GFP to microspheres has prevented GFP standards for flow cytometry from being developed.
Relatively small fluorophores such as fluorescein isothiocyanate have been attached to microbeads and used as calibration standards for flow cytometry. See, e.g., “Standardizing Flow Cytometry: Construction of a Standardized Fluorescence Calibration Plot Using Matching Spectral Calibrators” by A. Schwartz et al., Cytometry 26, 22 (1996), and “Model System Evaluating Fluorescein-Labeled Microbeads as Internal Standards to Calibrate Fluorescence Intensity on Flow Cytometers,” by Robert F. Vogt, Jr. et al., Cytometry 10, 294 (1989).
It is well known that biotin and the egg-white protein avidin (or streptavidin, its bacterial relative from Streptomyces avidinii), form a complex having a very large affinity (K
a
=10
15
M
−1
). This interaction is so strong that even biotin coupled to proteins is available for binding by avidin. See, e.g.,
Avidin-Biotin Technology,
(Eds. Meir Wilchek and Edward A. Bayer, Academic Press, Inc. (1990)), in Volume 184 of
Methods in Enzymology.
Accordingly, it is an object of the present invention to produce recombinants fluorescent protein microsphere calibration standards.
Another object of the present invention is to produce green fluorescent protein microsphere calibration standards.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the method for the preparation of recombinant fluorescent protein standard microspheres hereof may comprise in combination: fusing an affinity tag with the recombinant fluorescent protein; and attaching the affinity tag to a binding partner incorporated onto the microspheres, whereby the recombinant fluorescent protein is affixed to the microspheres.
Preferably, the recombinant fluorescent protein is enhanced green fluorescent protein, the affinity tag is biotin, and the binding partner is avidin or streptavidin.
It is also preferred that the microspheres are avidin or streptavidin microspheres.
REFERENCES:
patent: 5545551 (1996-08-01), Johnson et al.
patent: 5723218 (1998-03-01), Haugland et al.
patent: 5723584 (1998-03-01), Schatz
Patterson et al. (Nov. 1997) Biophys. J. Vol. 73, pp. 2782-2790.*
Oker-Blom et al. (1996) FEBS Letters 389, pp. 238-243.*
Cronan (1990) J. Biol. Chem. 265/18, pp. 10327-10333.*
Mitchell et al. (1995) FEBS Letters 368, pp. 148-150.*
Vogt et al. (1989) Cytometry, 10, pp. 294-302.*
George H. Patterson et al., “Use of the Green Fluorescent Protein and its Mutants in Quantitative Fluorescence Microscopy,” Biophysical Journal 73, 2782 (1997).
Martin Chalfie et al., “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263, 802 (1994).
A. Schwartz et al., “Standardizing Flow Cytometry: Construction of a Standardized Fluorescence Calibration Plot Using Matching Spectral Calibrators,” Cytometry 26, 22 (1996).
Robert F. Vogt, “Model System Evaluating Fluorescein-Labeled Microbeads as Internet Standards to Calibrate Fluorescence Intensity on Flow Cytometers,” Cytometry 10, 294 (1989).
Avidin-Biotin Technology, (Eds. Meir Wilchek and Edward A. Bayer, Academic Press, Inc. (1990), in vol. 184 of Methods in Enzymology.
John E. Cronan, Jr., “Biotination of Proteins in vivo,” J. Biolog. Chem. 265, 10327 (1990).
J. T. Murphy and J. C. Lagarias, “The Phytoflkuors: A New Class of Fluorescent Protein Probes,” Current Biology 7, 870 (1997).
Lehnert Bruce E.
Nolan John P.
Nolan Rhiannon L.
Ruscetti Teresa
Carlson Karen Cochrane
Freund Samuel M.
The Regents of the University of California
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