Note: Descriptions are shown in the official language in which they were submitted.
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RENILLA RENIFORMIS GREEN FLUORESCENT PROTEIN AND MUTANTS THEREOF
BACKGROUND OF THE INVENTION
The green fluorescent protein (GFP) from the jellyfish Aeqzco~ea victo~ia has
become an
extremely useful tool for tracking and quantifying biological entities in the
fields of
biochemistry, molecular and cell biology, and medical diagnostics (Chalfie et
al., 1994, Science
263: 802-805; Tsien, 1998, Ann, Rev. Biochem. 67: 509-544). There are no
cofactors or
substrates required for fluorescence, thus the protein can be used in a wide
variety of organisms
and cell types. GFP has been used as a reporter gene to study gene expression
in vivo by
insertion downstream of a test promoter. The protein has also been used to
study the subcellular
localization of a number of proteins by direct fusion of the test protein to
GFP, and GFP has
become the reporter of choice for monitoring the infection efficiency of viral
vectors both in cell
culture and in animals. In addition, a number of genetic modifications have
been made to GFP
resulting in variants for which spectral shifts correspond to changes in the
cellular environm~;~t
such as pH, ion flux, and the phosphorylation state of the cell. Perhaps the
most promising role
for GFP as a cellular indicator is its application to fluorescence resonance
energy transfer
(FRET) technology. FRET occurs with fluorophores for which the emission
spectrum of one
overlaps with the excitation spectrum of the second. When the fluorophores are
brought into
close proximity, excitation of the "donor" fluorophore results in emission
from the "acceptor".
Pairs of such fluorophores are thus useful for monitoring molecular
interactions. Fluorescent
proteins such as GFP or variants thereof are useful for analysis of
protein:protein interactions in
vivo or in vitro if their fluorescent emission and excitation spectra overlap
to allow FRET. The
donor and acceptor fluorescent proteins rnay be produced as fusions with the
proteins one wishes
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to analyze for interactions. These types of applications of GFPs are
particularly appealing for
high throughput analyses, since the readout is direct and independent of
subcellular localization.
Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs
blue light
with excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and
emits green
fluorescence with an emission wavelength of about 510 nm and a minor peak near
540 nm (Ward
' et al., 1979, Photochem. Photobiol. Rev. 4: 1-57). The excitation maximum of
A. victoria GFP
is not within the range of wavelengths of standard fluorescein detection
optics. Further, the
breadth of the excitation and emission spectra of the A. victoria GFP are not
well suited for use
in applications involving FRET. In order to be useful in FRET applications,
the excitation and
emission spectra of the fluorophores are preferably tall and narrow, rather
than low and broad.
There is a need in the art for GFP proteins that are amenable to the use of
standard fluorescein .
excitation and detection optics. There is also a need in the art for GFP
proteins -with nan-ow,
preferably non-overlapping spectral peaks.
The use of A. victoria GFP as a reporter fur gene expression studies, while
very popular,
is hindered by relatively Iow quantum yield (the brightness of a fluorophore
is determined as the
product of the extinction coefficient and the fluorescence quantum yield).
Generally, the A.
victoria GFP coding sequences must be linked to a strong promoter, such as the
CMV promoter
or strong exogenous regulators such as the tetracycline transactivator system,
in order'to produce
readily detectable signal. This makes it difficult to use GFP as a reporter
for examining the
activity of native promoters responsive to endogenous regulators. Higher
intensity would
obviously also increase the sensitivity of other applications of GFP
technology. There is a need
in the art for GFP proteins with higher quantum yield.
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Another disadvantage of A. victoria GFP involves fluctuations in its spectral
characteristics with changes in pH. At high pH (pH 11-12), the wild-type A.
victoria GFP loses
absorbance and excitation amplitude at 395 nm and gains amplitude at 470 nm
(Ward et al.,
1982, Photochem. Photobiol. 35: 803-808). A. victoria fluorescence is also
quenched at acid pH,
with a pica around 4.5. There is a need in the art for GFPs exhibiting
fluorescence that is less
sensitive to pH fluctuations.
Further, in order to be more useful in a broad range of applications, there is
a need in the
art for GFP proteins exhibiting increased stability of fluorescence
characteristics relative to A.
victoria GFP, with regard to organic solvents, detergents and proteases often
used in biological
studies. There is also a need in the art for GFP proteins that are more likely
to be soluble in a
wider range of cell types and less likely to interfere non-specifically with
endogenous proteins
than A. victoria GFP.
A number of modifications to A. victoria GFP have been made with the aim of
enhancing
the usefulness of the protein. For example, modifications aimed at enhancing
the brightness of
the fluorescence emissions or the spectral characteristics of either the
excitation or emission
spectra or both have been made. It is noted that the stated aim of several of
these modification
approaches was to make an A. victoria GFP that is more similar to R.
reniformis GFP in its
excitation and emission spectra and fluorescence intensity. .
Literature references relating to A. victoria mutants exhibiting altered
fluorescence
characteristics include, for example, the following. Heim et al. (1995, Nature
373: 663-664)
relates to mutations at S65 of A. victoria that enhance fluorescence intensity
of the polypeptide.
The S65T mutation to the A. victoria GFP is said to "ameliorate its main
problems and bring its
spectra much closer to that of Renilla".
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A review by Chalfie (1995, Photochem. Photobiol. 62: 651-656) notes that an
S65T
mutant of A. victoria, the most intensely fluorescent mutant of A, victoria
known at the time, is
not as intense as the R, reniformis GFP.
Further references relating to A. victoria mutants include, for example, Ehrig
et al., 1995,
FEBS Lett. 367: 163-166); Surpin et al., 1987, Photochem. Photobiol. 45
(Suppl):~ 955;
Delagrave et al., 1995, BioTechnology 13: 151-154; and Yang et al., 1996, Gene
173: 19-23.
Patent and patent application references relating to A. victoria GFP and
mutants thereof
include the following. U.S. Patent No. 5,874,304 discloses A. victoria GFP
mutants said to alter
spectral characteristics and fluorescence intensity of the polypeptide. U.S.
Patent No. 5,968,738
discloses A. victoria GFP mutants said to have altered spectral
characteristics. One mutation,
V163A, is said to result in increased fluorescence intensity. U.S. Patent No.
5,804,387 discloses
A. victoria mutants said to have increased fluorescence intensity,
particularly in response to
excitation with 488 nm laser light. U.S. Patent No. 5,625,048 discloses A.
victoria mutants said
to have altered spectral characteristics as well as several mutants said to
have increased
fluorescence intensity. Related U.S. Patent No. 5,7.77,079 discloses further
combinations of
mutations said to provide A. victoria GFP polypeptides with increased
fluorescence intensity.
International Patent Application (PCT) No. W098/21355 discloses A. victoria
GFP mutants said
to have increased fluorescence intensity, as do WO97/20078, W097/42320 and
W097/1I094.
PCT Application No. W098/06737 discloses mutants said to have altered spectral
characteristics, several of which are said to have increased fluorescence
intensity.
In addition to A. victoria, GFPs have been identified in a variety of other
coelenterates
and anthazoa, however only two GFPs have been cloned, those from A. victoy-ia
(Prasher, 1992,
Gene 111: 229-233) and from the sea pansy, Renilla miillef~i (WO 99/49019).
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SUMMARY OF THE INVENTION
The invention encompasses recombinant polynucleotides encoding the GFP from R.
reniformis, as well as polynucleotides encoding variants and fusion
polypeptides of R. reniformis
GFP, as well as methods of using such polynucleotides and polypeptides.
More particularly, the invention encompasses a recombinant polynucleotide
which
comprises the sequence of SEQ ID NO: 1.
In one embodiment, the recombinant polynucleotide which comprises the sequence
of
SEQ ID NO: 1 further comprises a sequence encoding at least one fused
heterologous
polypeptide domain.
The invention further encompasses a recombinant vector comprising a
polynucleotide
sequence encoding R. reniformis GFP.
In one embodiment, the sequence encoding R. reniformis GFP is SEQ ID NO: 1.
In another embodiment the recombinant vector is selected from the group
consisting of a
plasmid, a bacteriophage, a virus, and a retrovirus.
The invention further encompasses a cell comprising a recombinant
polynucleotide
encoding R. reniformis GFP.
The invention further encompasses a cell comprising a recombinant vector
comprising a
polynucleotide sequence encoding R. reniformis GFP, or the polynucleotide
sequence of SEQ ID
NO: 1.
The invention further encompasses an isolated recombinant polypeptide
comprising the
amino acid sequence of SEQ ID NO: 2.
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The invention further encompasses a recombinant polypeptide comprising the
amino acid
sequence of R. reniformis GFP or a variant thereof and at least one fused
heterologous
polypeptide domain.
In one embodiment, the at least one fused heterologous polypeptide domain is
fused to
the amino-terminal end of the R. reniformis GFP or variant thereof.
In another embodiment, the at least one fused heterologous polypeptide domain
is fused
to the carboxy-terminal end of the R. reniformis GFP or variant thereof.
In another embodiment, the at least one fused heterologous polypeptide domain
is fused
to the R. reniformis GFP or variant thereof via a linker sequence.
The invention further encompasses a method of producing R. reniformis GFP
comprising
the steps of: a) introducing a recombinant vector comprising a polynucleotide
sequence encoding
R. reniformis GFP to a cell; b) culturing the cell of step (a); and c)
isolating R.' reniformis GFP
from the cell.
In one ernbodimeTt, the cell is a bacterial cell.
In another embodiment, the cell is a eukaryotic cell.
In a preferred embodiment, the eukaryotic cell is selected from the group
consisting of
yeasts, insect cells, and mammalian cells. It is preferred that the mammalian
cells are human.
In another embodiment, the polynucleotide sequence is a humanized sequence.
The invention further encompasses a polynucleotide encoding an altered R.
reniformis
GFP polypeptide with increased fluorescence intensity relative to wild-type R.
reniformis GFP.
In one embodiment, the polypeptide has at least one mutation relative to wild
type R.
reniformis GFP in the stretch of amino acids defined by amino acids 64-69 of
SEQ ID NO: 2.
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The invention further encompasses a polynucleotide encoding an R. reniformis
GFP
polypeptide with an excitation spectrum that is detectably distinct from that
of wild-type R.
reniformis GFP.
The invention further encompasses a polynucleotide encoding an R. ~reniformis
GFP
polypeptide with an emission spectrum that is detectably distinct from that of
wild-type R.
reniformis GFP.
The invention further encompasses a method of detecting protein:proteiri
interactions, the
method comprising the following steps: a) providing a first fusion polypeptide
comprising a first
polypeptide domain and a first R. reniformis GFP-derived polypeptide, and a
second fusion
polypeptide comprising a second polypeptide domain and a second R. renifonnis
GFP-derived
polypeptide, wherein the emission spectrum of the first R. reniformis GFP-
derived polypeptide
overlaps the excitation spectrum of the second R. reniformis GFP-derived
polypeptide,._ the
second R. reniformis GFP-derived polypeptide emits fluorescence with a
spectrum that is
distinguishable from fluorescence emitted by the first R. Reniformis GFP-
derived polypeptide,
and wherein the first R. reniformis GFP-derived polypeptide may be excited by
a spectrum of
light that does not excite fluorescence emission by the second R. reniformis
GFP-derived
polypeptide; b) mixing the first and the second fusion polypeptides; c)
irradiating the mixture of
step (b) with a spectrum of light that excites the first R. reniformis GFP-
derived polypeptide to
emit fluorescence but does not excite the second R. reniformis GFP-derived
polypeptide; and
a,,
d) detecting fluorescence emission from the second R. reniformis GFP-derived
polypeptide,
wherein the fluorescence emission from the second R. reniformis GFP
polypeptide indicates
protein:protein interaction between the first and the second polypeptide
domains.
In one embodiment, the method is performed in a living cell.
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The invention further encompasses a method of determining the location of a
polypeptide
of interest in a cell, wherein a polynucleotide sequence encoding the
polypeptide of interest is
known, the method comprising the steps of: a) linking the polynucleotide
sequence encoding the
polypeptide of interest with a polynucleotide encoding R. reniformis GFP, such
that the linked
polynucleotide sequences are fused in frame; b) introducing the linked
polynucleotide sequences
to a cell; and c) determining the location of the polypeptide encoded by the
linked poly~-~ucleotide
sequences.
In one embodiment the method is performed in a living cell.
The invention further encompasses a method of identifying cells to which a
recombinant
vector has been introduced, the method comprising the steps of: a) introducing
a recombinant
vector to a population of cells, wherein the recombinant vector encodes R.
reniformis GFP; b)
illuminating the population with light within the excitation spectrum of R:
reriifonnis GFP;. and
c) detecting fluorescence in the emission spectrum of R. reniformis GFP in the
population,
thereby identifying a cell to which the recombinant vector has been
introduced.
In one embodiment, the GFP is expressed as a fusion polypeptide.
In another embodiment, the GFP is expressed as a distinct polypeptide.
In another embodiment, the cell is identified by FACS analysis. .
The invention further encompasses a method of monitoring the activity of a
transcriptional regulatory sequence, the method comprising the steps of a)
operably linking a
nucleic acid sequence comprising the transcriptional regulatory sequence to a
nucleic acid
sequence encoding R. reniformis GFP of SEQ ID NO: 2 to form a reporter
construct; b)
introducing the reporter construct to a cell; and c) detecting R. reniformis
GFP fluorescence in
the cell, wherein the fluorescence reflects the activity of the
transcriptional regulatory sequence.
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The invention further encompasses a method of detecting a modulator of a
transcriptional
regulatory sequence, the method comprising the steps of a) operably linking a
nucleic acid
sequence comprising the transcriptional regulatory sequence to a nucleic acid
sequence encoding
R. reniformis GFP of SEQ ID NO: 2 to form a reporter construct, wherein the
transcriptional
regulatory sequence is responsive to the presence of the modulator; b)
introducing the reporter
construct to a cell; and c) detecting R. reniformis GFP fluorescence in the
cell, wherein the
fluorescence indicates the presence of the modulator.
In one embodiment, the modulator is selected from the group consisting of a
hormone or
lipid soluble transcriptional modulator, a growth factor, and a heavy metal.
The invention further encompasses a method of screening for an inhibitor of a
transcriptional regulatory sequence, the method comprising the steps of a)
operably linking a
nucleic acid sequence comprising the transcriptional regulatory sequence to. a
nucleic acid
sequence encoding R. reniformis GFP of SEQ ID NO: 2 to form a reporter
construct; b)
introducing the reporter construct to a cell; c) contacting the cell with a
candidate inhibitor of the
transcriptional regulatory sequence; and d) detecting R. reniformis GFP
fluorescence in the cell,
wherein a decrease in the fluorescence relative to that detected in the
absence of the candidate
inhibitor indicates that the candidate inhibitor inhibits the activity of the
transcriptional
regulatory sequence. . .
The invention further encompasses a method of producing a fluorescent
molecular weight
marker, the method comprising the steps of a) linking a nucleic acid sequence
encoding R.
Reniformis GFP in frame to a nucleic acid sequence encoding a polypeptide of
known relative
molecular weight such that the linked molecules encode a fusion polypeptide;
b) introducing the
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linked nucleic acid sequences of (a) to a cell; c) isolating the fusion
polypeptide.from the cell,
wherein the fusion polypeptide is a molecular weight marker.
The invention further encompasses a polynucleotide encoding R. reniformis GFP
or a
variant of R. reniformis GFP, wherein the polynucleotide comprises at least
one humanized
codon sequence.
The invention further encompasses a humanized polynucleotide, the
polynucleotide
encoding R. reniformis GFP or a variant of R. reniformis GFP.
In one embodiment, the humanized polynucleotide comprises the sequence of SEQ
ID
NO: 3.
The invention further encompasses a recombinant vector comprising a humanized
R.
reniformis GFP polynucleotide.
The invention further encompasses a cell containing a recombinant vector
comprising a
humanized R. reniformis GFP polynucleotide.
A~ used herein, the term "R. reniformis green fluorescent protein" or"R.
reniformis
GFP" refers to a polypeptide of SEQ ID NO: 2 or to a fluorescent variant
thereof. An R.
reniformis GFP variant encompasses polypeptides of SEQ ID NO: 2 that bear one
or more
mutations, including insertion or deletion of one or more amino acids, either
at the N or C
termini of the polypeptide or internal to the coding sequence. Variants of R.
reniformis GFP
retain the ability to emit light when excited by light within a given part of
the spectrum, and may
be excited by light of, or emit light in a portion of the spectrum that
differs detectably from that
which excites or which is emitted by wild-type R. reniformis GFP of SEQ ID NO:
2. In addition
to variants exhibiting different excitation or emission spectra, R. reniformis
GFP variants include
variants exhibiting increased fluorescence intensity relative to wild-type R.
reniformis GFP.
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The term "variant thereof' when used in reference to an R. reniformis
polynucleotide
coding sequence means that the sequence bears one or more nucleotide
differences relative to the
sequence of the wild-type R. reniformis coding sequence. A variant of an R.
reniformis
polynucleotide sequence encodes an R. reniformis GFP polypeptide or a variant
thereof. A
variant of an R. reniformis polynucleotide coding sequence includes a
humanized polynucleotide
coding sequence. A variant polynucleotide directs the expression of an amount
of fluorescent
polypeptide at least equal to, or greater than, the amount expressed from an
equal mass amount
or from an equal number of copies of a non-humanized R. reniformis GFP
polynucleotide
sequence.
The term "humanized polynucleotide" or "humanized sequence" refers to a
polynucleotide coding sequence in which one or more, including 5 or more, 10
or more, 20 or
more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 200 or
more, or even all
colons of the polynucleotide coding sequence for a non-human polypeptide
(i.e., a polypeptide
not naturally expressed in humans) have been altered to a colon sequence more
preferred for
expression in human cells. Because there are 64 possible combinations of the 4
DNA nucleotides
in colon groups of 3, the genetic code is redundant for many of the 20 amino
acids. Each of the
different colons for a given amino acid encodes the incorporation of that
amino acid into a
polypeptide. However, within a given species there tends to be a preference
for certain of the
redundant colons to encode a given amino acid. The "colon preference" of R.
reniformis is
different from that of humans (this colon preference is usually based upon
differences in the
level of expression of the tRNAs containing the corresponding anticodon
sequences). In order to
obtain high expression of a non-human gene product in human cells, it is
advantageous to
change one or more non-preferred codorts to a colon sequence that is preferred
in human cells.
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Table 1 shows the preferred codons for human gene expression. A codon sequence
is preferred
for human expression if it occurs to the left of a given codon sequence in the
table. Optimally,
but not necessarily, less preferred codons in a non-human polynucleotide
coding sequence are
humanized by altering them to the codon most preferred for that amino acid in
human gene
expression. The amount of fluorescent polypeptide expressed in a human cell
from a humanized
GFP polynucleotide sequence is at least two-fold greater, on either a mass or
a fluorescence
intensity scale per cell, than the amount expressed from an equal amount or
number of copies of
a non-humanized GFP polynucleotide.
As used herein, the term "humanized codon" means a codon sequence, within a
polynucleotide sequence encoding a non-human polypeptide, that has been
changed to a codon
sequence that is more preferred for expression in human cells relative to that
codon encoded by
the non-human organism from which the non-human polypeptide is derived:
Species-specific
codon preferences stem in part from differences in the expression of tRNA
molecules with the
appropriate anticodon sequence. That is, one factor in the species-specific
codon preference is
the realtionship between a codon and the amount of corresponding anticodon
tRNA expressed.
It should be understood that any of the recombinant vectors of the invention
may
comprise a humanized polynucleotide encoding R. reniformis GFP or a variant
thereof.
Similarly, any of the cells of the invention may comprise vectors comprising a
humanized
polynucleotide encoding R. reniformis GFP or a variant thereof. It should also
be understood
that all claimed methods using polynucleotides encoding R. reniformis GFP may
be performed
with humanized polynucleotides encoding R. reniformis GFP or variants of R.
reniformis GFP.
Finally, any R. reniformis GFP polypeptide of the invention may be expressed
from a humanized
R. reniformis GFP polynucleotide coding sequence.
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As used herein, the term "wild-type R. reniformis GFP" refers to a polypeptide
of SEQ
ID NO: 2.
As used herein, the term "increased fluorescence intensity" or "increased
brightness"
refers to fluorescence intensity or brightness that is greater than that
exhibited ~by wild-type R.
reniformis GFP under a given set of conditions. Generally, an increase in
fluorescence intensity
or brightness means that fluorescence of a variant is at least 5% or more, and
preferably 10%,
20%, 50%, 75%, 100% or more, up to even 5 times, 10 times, 20 times, 50 times
or 100 times or
more intense or bright than wild-type R. reniformis GFP under a given set of
conditions.
As used herein, the term "fused heterologous polypeptide domain" refers to an
amino
acid sequence of two or more amino acids fused in frame to R. reniformis GFP
or a variant
thereof A fused heterologous domain may be linked to the N or C terminus of
the R. reniformis
GFP polypeptide or variant thereof.
As used herein, the term "fused to the amino-terminal end" refers to the
linkage of a
polypeptide sequence to the amino terminus of another polypeptide. The linkage
may be direct
or may be mediated by a short (e.g., about 2-20 amino acids) linker peptide.
As used herein, the term "fused to the carboxy-terminal end" refers to the
linkage of a
polypeptide sequence to the carboxyl terminus of another polypeptide. The
linkage may be
direct or may be mediated by a linker peptide.
As used herein, the term "linker sequence" refers to a short (e.g., about 1-20
amino acids)
sequence of amino acids' that is not part of the sequence of either of two
polypeptides being
joined. A linker sequence is attached on its amino-terminal end to one
polypeptide or
polypeptide domain and on its carboxyl-terminal end to another polypeptide or
polypeptide
domain.
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As used herein, the term "excitation spectrum" refers to the wavelength or
wavelengths
of light that, when absorbed by a fluorescent polypeptide molecule of the
invention, causes
fluorescent emission by that molecule.
As used herein, the term "emission spectrum" refers to the wavelength~.or
wavelengths of
light emitted by a fluorescent polypeptide.
As used herein, the terms "distinguishable" or "detestably distinct" mean that
standard
filter sets allow either the excitation of one form of a polypeptide without
excitation of another
given polypeptide, or similarly, that standard filter sets allow the
distinction of the emission from
one polypeptide form from the emission spectrum of another. Generally,
distinguishable or
detestably distinct excitation or emission spectra have peaks that vary by
more than 1 nm, and
preferably vary by more than 2, 3, 4, 5, 10 or more nm.
As used herein, the term "fusion polypeptide" refers to a polypeptide that. is
comprised of
two or more amino acid sequences, from two or more proteins that are not found
linked in
nature, that are physically linked by a peptide bond.
As used herein, the term "emission spectrum overlaps the excitation spectrum"
means
that light emitted by one fluorescent polypeptide is of a wavelength or
wavelengths that causes
excitation and emission by another fluorescent polypeptide.
As used herein, the term "population of cells" refers to a plurality of cells,
preferably, but
not necessarily of same type or strain.
As used herein the term "distinct polypeptide" refers to a polypeptide that is
not
expressed as a fusion polypeptide.
As used herein, the term "FACS analysis " refers to the method of sorting
cells,
fluorescence activated cell sorting, wherein cells are stained with or express
one or more
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fluorescent markers. In this method, Bells are passed through an apparatus
that excites and
detects fluorescence from the marker(s). Upon detection of fluorescence in a
given portion of the
spectrum by a cell, the FAGS apparatus allows the separation of that cell from
those not
expressing that fluorescence spectrum.
As used herein, the term "lipid soluble transcriptional modulator" refers to a
composition
that is capable of passing through cell membranes (nuclear or cytoplasmic) and
has a positive or
negative effect on the transcription of one or more genes or constructs.
As used herein, the term "operably linked" means that a given coding sequence
is joined
to a given transcriptional regulatory sequence such that transcription of the
coding sequence
occurs and is regulated by the regulatory sequence.
As used herein, the teen "reporter construct" refers to a polynucleotide
construct
encoding a detectable molecule, linked to a transcriptional regulatory
sequence , conferring
regulated transcription upon the polynucleotide encoding the detectable
molecule. A detectable
molecule is preferably an R. reniformis GFP or variant thereof.
As used herein, the term "responsive to the presence of a modulator" means
that a given
transcriptional regulatory sequence is either turned on or turned off in the
presence of a given
compound. As used herein, gene expression is "turned on" when the polypeptide
encoded by the
gene sequence (e.g., a GFP polypeptide or variant thereof) is detectable over
background, or
alternatively, when the polypeptide is detectable in an increased amount over
the amount
detected in the absence of a given modulator compound. In this context,
"increased amount"
means at least 10%, preferably 20%, 50%, 75%, 100% or more, up to even 5
times, 10 times, 20
times, 50 times, or 100 times or more higher than background detection, with
background
detection being the amount of signal observed in the absence of the modulator
compound.
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As used herein, the term "modulator of a transcriptional regulatory sequence"
refers to a
compound or chemical moiety that causes a change in the level of expression
from a
transcriptional regulatory sequence. Preferably, the change is detectable as
an increase or
decrease in the detection of a reporter molecule or reporter molecule
activity, with at least 10%,
20%, 50%, 75%, 100%, or even 5 times, 10 times, 20 times, 50 times or 100
times or more
increased or decreased level of reporter signal relative to the absence of a
given modulator.
As used herein the term "inhibitor of a transcriptional regulatory sequence"
refers to a
compound or chemical moiety that causes a decrease in the amount of a reporter
molecule or
reporter molecule activity expressed from a given transcriptional regulatory
sequence. As used
herein, the term "decrease" when used in reference to the detection of a
reporter molecule or
reporter molecule activity means that detectable activity is reduced by at
least 10%, 20%, 50%,
75%, or even 100% (i.e., no expression), relative to the amount detected~in
the absence of a
given compound or chemical moiety. As used herein the term "candidate
inhibitor" refers to a
compound or chemical moiety being tested for inhibitory activity in an assay.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the coding sequence of R. reniformis GFP, SEQ ID NO: 1.
Figure 2 shows the amino acid sequence of R. reniformis GFP, SEQ ID NO: 2.
Figure 3 is a graphical representation of R. reniformis GFP expressed in
transduced cells.
The unshaded peak represents the uninfected cell population; the shaded peak
represents cells
transduced with the GFP-expressing virus. In this experiment, 44% of the
transduced population
showed fluorescence above background.
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Figure 4 shows fluorescence spectra of recombinant R. reniformis GFP. Spectra
were
measured using 10 nm bandwidths. The y-axis scales for the two peaks have been
normalized so
that the fluorescence profiles have equal amplitude.
Figure 5 shows the sequence of a humanized R. reniformis GFP polynucleotide
sequence
(SEQ ID NO: 3).
Figure 6 shows a sequence alignment between non-humanized and humanized R.
~-ercifof°~ris GFP. Vertical lines represent homology between the
humanized and non-humanized
genes. Gaps represent nucleotides that were altered to produce the hrGFP gene.
Figure 7 shows the relative fluorescence of CHO cells transduced by retroviral
vectors
harboring non-humanized or humanized R. f°eyiiforfyZis GFP. Cells were
infected with undiluted
supernatants containing virus derived from the two GFP vectors, or media alone
(No Virus).
Figure 8 shows the relative fluorescence of 293 cells harboring single copy
proviral
integrants from which either rGFP, hrGFP or EGFP is expressed. The % UR value
indicates the
number of cells which fluoresce above background. The raw % UR for the "No
Virus" control
was 0.15%, and was subtracted from the values for all cell populations.
DESCRIPTION
The invention relates to the GFP from R. reniformis. Polynucleotide sequences
encoding
the R. reniformis GFP are disclosed herein, as are polypeptide sequences for
R. reniformis GFP
and variants thereof.
R. reniformis GFP polynucleotides were isolated through PCR amplification
using an R.
reniformis cDNA library prepared in lambda phage. Full length coding sequences
were isolated,
sequenced, and inserted into a variety of different expression vectors.
17
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Also disclosed herein are methods of producing R, reniformis GFP polypeptides
or
variants thereof, the methods comprising introducing an expression vector
encoding R.
reniformis GFP or a variant thereof into a cell, culturing the cell, and
isolating the GFP
polypeptides or variants.
I. How to Make R. reniforrnis GFP Polynucleotides and Polypeptides According
to the
Invention.
A number of methodologies were combined to provide the invention disclosed
herein,
including molecular, cellular and biochemical approaches. Polynucleotides
encoding R.
reniformis GFP are obtained in any of several different ways, including direct
chemical
synthesis, library screening and PCR amplification. R. reniformis GFP
polypeptides are
obtained by expression from recombinant polynucleotide sequences in
appropriate organisms.
Useful variants of R. reniformis GFP polypeptides are produced in similar ways
following the
introduction of mutations to the polynucleotide sequence encoding wild-type R.
reniformis GFP.
Those methodologies necessary to make and use the R. reniformis GFP
polynucleotides,
polypeptides and variants thereof of the invention are discussed in detail
below.
A. Isolation of R. reniformis GFP-encoding polynucleotide sequences.
1. R. reniformis cDNA Library Preparation.
Construction methods for libraries in a variety of different vectors,
including, for
example, bacteriophage, plasmids, and viruses capable of infecting eukaryotic
cells are well
known in the art. Any known library production method resulting in largely
full-length clones of
expressed genes may be used to provide a template for the isolation of GFP-
encoding
polynucleotides from R. reniformis.
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WO 01/64843 PCT/USO1/06131
For the library used to isolate the GFP-encoding polynucleotides disclosed
herein, the
following method was used. Poly(A) RNA was prepared from R.
~°ef2iforsnis organisms as
described by Chomczynski, P. and Sacchi, N. (1987, Anal. Biochem. 162: 156-
159). cDNA was
prepared using the ZAP-cDNA Synthesis K.it (Stratagene cat.# 200400) according
to the
manufacturer's recommended protocols and inserted between the EcoR I and Xho I
sites in the
vector Lambda ZAP II. The resulting library contained 5 x 106 individual
primary clones, with
an insert size range of 0.5 - 3.0 kb and an average insert size of 1.2, kb.
The library was
amplified once prior to use as template fox PCR reactions.
2. Isolation of R. reniformis GFP Coding Sequence by PCR.
The R. reniformis GFP coding sequence was isolated by polymerise chain
reaction
(PCR) amplification of the sequence from within the cDNA library described
herein. A large
number of PCR methods are known to those skilled in the art. Thermal-cycled
PCR (Mullis~ and
Faloona, 1987, Methods Enzymol., 155: 335-350; see also, PCR Protocols, 1990,
Academic
Press, San Diego, CA, USA for a review of PCR methods) uses multiple cycles of
DNA
replication catalyzed by a thermostable, DNA-dependent DNA polymerise to
amplify the target
sequence of interest. Briefly, oligonucleatide primers are selected such that
they anneal on either
side and on opposite strands of a sequence to be amplified. The primers are
annealed and
extended using a template-dependent thermostable DNA polymerise, followed by
thermal
denaturation and annealing of primers to both the original template sequence
and the newly-
extended template sequences, after which primer extension is performed.
Repeating such cycles
results in exponential amplification of the sequences between the two primers.
In addition to thermal cycled PCR, there are a number of other nucleic acid
sequence
amplification methods that may be used to amplify, and isolate a GFP-encoding
polypeptide
19
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WO 01/64843 PCT/USO1/06131
according to the invention from an R. reniformis cDNA library. These include,
for example,
isothermal 3SR (Gingeras et al:, 1990, Annales de Biologie Clinique, 48(7):
498-501; Guatelli et
al., 1990, Proc. Natl. Acad. Sci. U.S.A., 87: 1874), and the DNA ligase
amplification reaction
(LAR), which permits the exponential increase of specific short sequences
through the activities
of any one of several bacterial DNA Iigases (Wu and Wallace, 1989, Genomics,
4: 560). The
contents of both of these references are incorporated herein in their entirety
by reference.
To amplify a sequence encoding R. reniformis GFP from an R. reniforrriis cDNA
library,
the following approach was taken. The R. resaifoj°mis GFP coding
sequence was amplified using
the 5' primer 5'-AATTATTAGAATTCACCATGGTGAGTAAACAAATATTGAAGAAC-3'
and the 3' primer 5'-ATAATATTCTCGAGTTAAACCCATTCGTGTAAGGATCC-3. The 5'
primer contains an EcoR I recognition site to facilitate subsequent cloning of
the amplified
fragment, followed by the Kozak consensus translation initiation sequence
ACCATGG. The 3'
primer contains an Xho I recognition site to facilitate cloning of the
amplified fragment.
Oligonucleotides may be purchased from any of a number of commercial suppliers
(for example,
Life Technologies, Inc., Operon Technologies, etc.). Alternatively,
oligonucleotide primers may
be synthesized using methods well known in the art , including, for example,
the phosphotriester
(see Narang, S.A., et al., 1979, Meth. Enzymol., 68:90; and U.S. Pat. No.
4,356,270),
phosphodiester (Brown, et al., 1979, Meth. Enzymol., 68:109), and
phosphoramidite
(Beaucage, 1993, Meth. Mol. Biol., 20:33) approaches. Each of these references
is
incorporated herein in its entirety by reference.
PCR was carned out in a 50 ~I reaction volume containing lx TaqPlus Precision
buffer
(Stratagene), 250 ACM of each dNTP, 200 nM of each PCR primer, 2.5 U TaqPlus
Precision
CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
enzyme (Stratagene) and approximately 3 x 10~ lambda phage particles from the
amplified
cDNA library described above. Reactions were carried out in a Robocycler
Gradient 40
(Stratagene) as follows: 1 min at 95 °C (1 cycle), 1 min at 95
°C, 1 rnin at 53 °C, 1 min at 72 °C
(40 cycles), and 1 min at 72 °C ( 1 cycle). Reaction products were
resolved on ~a 1 % agarose gel,
and a band of approximately 700 by was excised and purified using the
StrataPrep DNA Gel
Extraction I~it (Stratagene). Other methods of isolating and purifying
amplified nucleic acid
fragments are well known to those skilled in the art. The PCR fragment was
subcloned by
digestion to completion with EcoRI and XhoI and insertion into the retroviral
expression vector
pFB (Stratagene) to create the vector pFB-rGFP. Both strands of the cloned GFP
fragment were
completely sequenced. The coding polynucleotide and amino acid sequences are
presented in
Figures 1 and 2, respectively. The R, reniformis and R. mulleri GFP coding
sequences are 83
homologous, and the proteins share 88% identical amino acid sequence. .
3. Isolation of R. reniformis GFP-encoding polynucleotides by library
screening.
An alternative method of isolating GFP-encoding polynucleotides according to
the
invention involves the screening of an expression library, such as a lambda
phage expression
library, for clones exhibiting fluorescence within the emission spectrum of
GFP when
illuminated with light within the excitation spectrum of GFP. In this way
clones may be directly
identified from within a large pool. Standard methods for plating lambda
phage, expression
libraries and inducing expression of polypeptides encoded by the inserts are
well established in
the art. Screening by fluorescence excitation and emission is carried out as
described herein
below using either a spectrofluorometer or even visual identification of
fluorescing plaques.
With either method, fluorescent plaques are picked and used to re-infect fresh
cultures one or
21
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WO 01/64843 PCT/USO1/06131
more times to provide pure cultures, from which GFP insert sequences may be
determined and
sub-cloned.
As another alternative, if a sequence is available for the polynucleotide one
wishes to
obtain, the polynucleotide may be chemically synthesized by one of skill in
the art. The same
synthetic methods used for the preparation of oligonucleotide primers
(described above) may be
used to synthesize gene coding sequences for GFPs of the invention. Generally
this would be
performed by synthesizing several shorter sequences (about 100 nt or less),
followed by
annealing and ligation to produce the full length coding sequence.
B. Production of R. reniformis GFP polypeptides and variants thereof.
The production of R. reniformis GFP polypeptides (e.g., the polypeptide with
the amino
acid sequence of SEQ ID NO: 2) and variants thereof from recombinant vectors
comprising
GFP-encoding polynucleotides of the invention may be effected in a number of
ways -known to
those skilled in the art. For example, plasmids, bacteriophage or viruses may
be introduced to
prokaryotic or eukaryotic cells by any of a number of ways known to those
skilled in the art.
Following introduction of R. renifonnis GFP-encoding polynucleotides to a
prokaryotic or
eukaryotic cell, expressed GFP polypeptides may be isolated using methods
known in the art or
described herein below. Useful vectors, cells, methods of introducing vectors
to cells and
methods of detecting and isolating GFP polypeptides. and variants thereof are
also described
herein below.
1. Vectors Useful According to the Invention.
There is a wide array of vectors known and available in the art that are
useful for the
expression of GFP polypeptides or variants thereof according to the invention.
The selection of a
particular vector clearly depends upon the intended use of the GFP polypeptide
or variant
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WO 01/64843 PCT/USO1/06131
thereof. For example, the selected vector must be capable of driving
expression of the
polypeptide in the desired cell type, whether that cell type be prokaryotic or
eukaryotic. Many
vectors comprise sequences allowing both prokaryotic vector replication and.
eukaryotic
expression of operably linked gene sequences.
Vectors useful according to the invention may be autonomously replicating,
that is, the
vector, for example, a plasmid, exists extrachromosomally and its replication
is not necessarily
directly linked to the replication of the host cell's genome. Alternatively,
the replication of the
vector may be linked to the replication of the host's chromosomal DNA, for
example, the vector
may be integrated into the chromosome of the host cell as achieved by
retroviral vectors.
Vectors useful according to the invention preferably comprise sequences
operably linked
to the GFP coding sequences that permit the transcription and translation of
the GFP sequence.
Sequences that permit the transcription of the linked GFP sequence include 'a
promoter and
optionally also include an enhancer element or elements permitting the strong
expression of the
linked sequences. The term "transcriptional regulatory sequences" refers to
the combination of a
promoter and any additional sequences conferring desired expression
characteristics (e.g., high
level expression, inducible expression, tissue- or cell-type-specific
expression) on an operably
linked nucleic acid sequence.
The selected promoter may be any DNA sequence that exhibits transcriptional
activity in
the selected host cell, and may be derived from a gene normally expressed in
the host cell or
from a gene normally expressed in other cells or organisms. Examples of
promoters include, but
are not limited to the following: A) prokaryotic promoters - E. coli lac, tac,
or trp promoters,
lambda phage PR or P~ promoters, bacteriophage T7, T3, Sp6 promoters, B.
subtilis alkaline
protease promoter, and the B. stearothermophilus maltogenic amylase promoter,
etc.; B)
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WO 01/64843 PCT/USO1/06131
eukaryotic promoters - yeast promoters, such as GAL1, GAL4 and other
glycolytic gene
promoters (see for example, Hitzeman et al., 1980, J. Biol. Chem. 255: 12073-
12080; Alber &
Kawasaki, 1982, J. Mol. Appl. Gen. 1: 419-434), LEU2 promoter (Martinez-Garcia
et al., 1989,
Mol Gen Genet. 217: 464-470), alcohol dehydrogenase gene pronnoters (Young et
al., 1982, in
Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., eds.,
Plenum Press,
NY), or the TPIl promoter (U.S. Pat. No. 4,599,311); insect promoters, such as
the polyhedrin
promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., 1992, FEBS Lett. 311: 7-
11); the P10
promoter (Vlak et al., 1988, J. Gen. Virol. 69: 765-776), the Autographs
californica polyhedrosis
virus basic protein promoter (EP 397485), the baculovirus immediate-early gene
promoter gene
1 promoter (U.S. Pat. Nos. 5,155,037 and 5,162,222), the baculovirus 39I~
delayed-early gene
promoter (also U.S. Pat. Nos. 5,155,037 and 5,162,222) and the OpMNPV
immediate early
promoter 2; mammalian promoters - the SV40 promoter (Subramani et aL, 1981,
Mol. Cell. Biol.
I: 854-864), metallothionein promoter (MT-I; Palmiter et al., 1983, Science
222: 809-814),
adenovirus 2 major late promoter (Yu et a1.,1984, Nucl. Acids Res. 12: 9309-
21),
cytomegalovirus (CMV) or other viral promoter (Tong et al., 1998, Anticancer
Res. 18:
719-725), or even the endogenous promoter of a gene of interest in a
particular cell type.
A selected promoter may also be linked to sequences rendering it inducible or
tissue-
specific. For example, the addition of a tissue-specific enhancer element
upstream of a selected
promoter may render the promoter more active in a given tissue or cell type.
Alternatively, or in
addition, inducible expression may be achieved by linking the promoter to any
of a number of
sequence elements permitting induction by, for example, thermal changes
(temperature
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CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
sensitive), chemical treatment (for example, metal ion- or IPTG-inducible), or
the addition of an
antibiotic inducing agent (for example, tetracycline).
Regulatable expression is achieved using, for example, expression systems that
are drug
inducible (e.g., tetracycline, rapamycin or hormone-inducible). Drug-
regulatable promoters that
are particularly well suited for use in mammalian cells include the
tetracycline regulatable
promoters, and glucocorticoid steroid-, sex hormone steroid-, ecdysone-,
lipopolysaccharide
(LPS)- and isopropylthiogalactoside (IPTG)-regulatable promoters. A
regulatable expression
system for use in mammalian cells should ideally, but not necessarily, involve
a transcriptional
regulator that binds (or fails to bind) nonmammalian DNA motifs in response to
a regulatory
agent, and a regulatory sequence that is responsive only to this
transcriptional regulator.
One inducible expression system that is well suited for the regulated
expression of a GFP
polypeptide of the invention or variant thereof, is the tetracycline-
regulatable expression system,
which is founded on the efficiency of the tetracycline resistance operon of E.
coli. The binding
constant between tetracycline and the tet repressor is high while the toxicity
of tetracycline for
mammalian cells is low, thereby allowing for regulation of the system by
tetracycline
concentrations in eukaryotic cell culture or within a mammal that do not
affect cellular growth
rates or morphology. Binding of the.tet repressor to the operator occurs with
high specificity.
Versions of the tet-regulatable system exist that allow either positive or
negative
regulation of gene expression by tetracycline. In the absence of tetracycline
or a tetracycline
analog, the wild-type bacterial tet repressor protein causes negative
regulation of genes driven by
promoters containing repressor binding elements from the tet operator
sequences. Gossen &
Bujard (1995, Science 268: 1766-1769; also International patent application
No. WO 96/01313)
CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
describe a tet-regulatable expression system that exploits this positive
regulation by tetracycline.
In this system, tetracycline binds to a tet repressor fusion protein, rtTA,
and prevents it from
binding to the tet operator DNA sequence, thus allowing transcription and
expression. of the
linked gene only in the presence of the drug.
This positive tetracycline-regulatable system provides one means of stringent
temporal
regulation of the GFP polypeptide of the invention or variant thereof (Gossen
& Bujard, 1995,
supra). ~ The tet operator. (tet O) sequence is now well known to those
skilled in the art. For a
review, the reader is referred to Hillen & Wissmann (1989) in Protein-Nucleic
Acid Interaction,
"Topics in Molecular and Structural Biology", eds. Saenger & Heinemann,
(Macmillan,
London), Vol. 10, pp 143-162. Typically the nucleic acid sequence encoding the
GFP
polypeptide is placed downstream of a plurality of tet O sequences: generally
5 to 10 such tet O
sequences are used, in direct repeats.
In addition to the tetracycline-regulatable systems, a number of other options
exist for the
regulated or inducible expression of a GFP polypeptide or variant thereof
according to the
invention. For example, the E. coli lac promoter is responsive to lac
repressor (lacI) DNA
binding at the lac operator sequence. The elements of the operator system are
functional in
heterologous contexts, and the inhibition of lacI binding to the lac operator
by IPTG is widely
used to provide inducible expression in both prokaryotic, and more recently,
eukaryotic cell
systems. In addition, the rapamycin-controlled transcriptional activator
system described by
Rivera et al. (1996, Nature Med. 2: 1028-1032) provides transcriptional
activation dependent on
rapamycin. That system has low baseline expression and a high induction ratio.
26
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WO 01/64843 PCT/USO1/06131
Another option for regulated or inducible expression of a GFP polypeptide or
variant
thereof involves the use of a heat-responsive promoter. Activation is induced
by incubation of
cells, transfected with a GFP construct regulated by a temperature-sensitive
transactivator, at the
permissive temperature prior to administration. For example,
transcription~regulated by a co-
transfected, temperature sensitive transcription factor active only at
37°C may be used if cells are
first grown at, for example, 32°C, and then switched to 37°C to
induce expression.
Tissue-specific promoters may' also be used to advantage in GFP-encoding
constructs of
the invention. A wide variety of tissue-specific promoters is known. As used
herein, the term
"tissue-specific" means that a given promoter is transcriptionally active
(i.e., directs the
expression of linked sequences sufficient to permit detection of the
polypeptide product of the
promoter) in less than all cells or tissues of an organism. A tissue specific
prarnoter is preferably
active in only one cell type, but may, fox example, be active in a particular
class or lineage of cell
types (e.g., hematopoietic cells). A tissue specific promoter useful according
to the invention
comprises those sequences necessary and sufficient for the expression of an
operably linked
nucleic acid sequence in a manner or pattern that is essentially the same as
the manner or pattern
of expression of the gene linked to that promoter in nature. The following is
a non-exclusive list
of tissue specific promoters and literature references containing the
necessary sequences to
achieve expression characteristic of those promoters in their respective
tissues; the entire content
of each of these literature references is incozporated herein by reference.
Examples of tissue
specific promoters useful with the R. Reniformis GFP of the invention are as
follows:
Bowman et al., 1995 Proc. Natl. Acad. Sci. USA 92,12115-12119 describe a brain-
specific
transfernn promoter; the synapsin I promoter is neuron specific (Schoch et
al., 1996 J. Biol.
27
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WO 01/64843 PCT/USO1/06131
Chem. 271, 3317-3323); the necdin promoter is post-mitotic neuron specific
(Uetsuki et al., 1996
J. Biol. Chem. 271, 918-924); 'the neurofilament light promoter is neuron
specific (Charron et al.,
1995 J. Biol. Chem. 270, 30604-30610); the acetylcholine receptor promoter is
neuron specific
(Wood et al., 1995 J. Biol. Chem. 270, 30933-30940); the potassium channel
promoter is high-
frequency firing neuron specific (Gan et al., 1996 J. Biol. Chem 271, 5859-
5865); the
chromogranin A promoter is neuroendocrine cell specific (Wu et al.; 1995 A.J.
Clin. Invest. 96,
568-578); the Von Willebrand factor promoter is brain endothelium specific
(Aird et al., 1995
Proc. Natl. Acad. Sci. USA 92, 4567-4571); the flt-1 promoter is endothelium
specific (Morishita
et al., 1995 J. Biol. Chem. 270, 27948-27953); the preproendothelin-1 promoter
is endothelium,
epithelium and muscle specific (Harats et al., 1995 J. Clin. Invest. 95, 1335-
1344); the GLUT4
promoter is skeletal muscle specific (Olson and Pessin, 1995 J. Biol. Chem.
270, 23491-23495);
the Slow/fast troponins promoter is slow/fast twitch myofibre specific (Corm
et al., 1995 Proc.
Natl. Acad. Sci. USA 92, 6185-6189); the -Actin promoter is smooth muscle
specific (Shimizu
et al., 1995 J. Biol. Chem. 270, 7631-7643); the Myosin heavy chain promoter
is smooth muscle
specific (Kallmeier et al., 1995 J. Biol. Chem. 270, 30949-30957); the E-
cadherin promoter is
epithelium specific (Hennig et al., 1996 J. Biol. Chem. 271, 595-602); the
cytokeratins promoter
is keratinocyte specific (Alexander et al., 1995 B. Hum. Mol. Genet. 4, 993-
999); the
transglutaminase 3 promoter is keratinocyte specific (J. Lee et al., 1996 J.
Biol. Chem. 271,
4561-4568); the bullous pemphigoid antigen promoter is basal keratinocyte
specific (Tamai et
al., 1995 J. Biol. Chem. 270, 7609-7614); the keratin 6 promoter is
proliferating epidermis
specific (Ramirez et al., 1995 Proc. Natl. Acad. Sci, USA 92, 4783-4787); the
collagen 1
promoter is hepatic stellate cell and skin/tendon fibroblast specific (Houglum
et al., 1995 J. Clin.
Invest. 96, 2269-2276); the type X collagen promoter is hypertrophic
chondrocyte specific (Long
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WO 01/64843 PCT/USO1/06131
& Linsenmayer, 1995 Hum. Gene Ther. 6, 419-428); the Factor VII promoter is
liver specific
(Greenberg et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12347-1235); the fatty
acid synthase
promoter is liver and adipose tissue specific (Soncini et al., 1995 J. Biol.
Chem. 270, 30339-
3034); the carbamoyl phosphate synthetase I promoter is portal vein hepatocyte
and small
intestine specific (Christoffels et aL, 1995 J. Biol. Chem. 270, 24932-24940);
the Na-K-Cl
transporter promoter is kidney (loop of Henle) specific (Igarashi et al., 1996
J. Biol. Chem. 271,
9666-9674); the scavenger receptor A promoter is macrophages and foam cell
specific (Horvai et
al., 1995 Proc. Natl. Acad. Sci. USA 92, 5391-5395); the glycoprotein IIb
promoter is
megakaryocyte and platelet specific (Block & Poncz, 1995 Stem Cells 13, 135-
145); the yc chain
promoter is hematopoietic cell specific (Markiewicz et al., 1996 J. Biol.
Chem. 271, 14849-
14855); and the CDl 1b promoter is mature myeloid cell specific (Dziennis et
al., 1995 Blood 85,
319-329).
Any tissue specific transcriptional regulatory sequence known in the art may
be used to
advantage with a vector encoding R. reniformis GFP or a variant thereof.
In addition to promoter/enhancer elements, vectors useful according to the
invention may
further comprise a suitable terminator. Such terminators include, for example,
the human growth
hormone terminator (Palmiter et al., 1983, supra), or, for yeast or fungal
hosts, the TPII (Alber &
Kawasaki, 1982, supra) or ADH3 terminator (McKnight et al., 1985, EMBO J. 4:
2093-2099).
Vectors useful according to the invention may also comprise polyadenylation
sequences
(e.g., the SV40 or AdSElb poly(A) sequence), and translational enhancer
sequences (e.g., those
from Adenovirus VA RNAs). Further, a vector useful according to the invention
may encode a
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signal sequence directing the recombinant polypeptide to a particular cellular
compartment or,
alternatively, may encode a signal directing secretion of the recombinant
polypeptide.
Coordinate expression of different genes from the same promoter in a
recombinant vector
maybe achieved by using an IRES element, such as the internal ribosomal entiy
site of Poliovirus
type 1 from pSBC-1 (Dirks et al., 1993, Gene 128:247-9). Internal ribosome
binding site (IRES)
elements are used to create multigenic or polycistronic messages. IRES
elements axe able to
bypass the ribosome scanning mechanism of 5' methylated Cap-dependent
translation and begin
translation at internal sites (Pelletier and Sonenberg, 1988, Nature 334: 320-
325). IRES elements
from two members of the picanovirus family (polio and encephalomyocarditis)
have been
described (Pelletier and Sonenberg, 1988, supra), as well an IRES from a
mammalian message
(Macejak and Sarnow, 1991 Nature 353: 90-94). Any of the foregoing may be used
in an R.
reniformis GFP vector in accordance with the present invention.
IRES elements can be linked to heterologous open reading frames. Multiple open
reading
frames can be transcribed together, each separated by an IRES, creating
polycistronic messages.
By virtue of the IRES element, each open reading frame is accessible to
ribosomes for efficient
translation. In this manner, multiple genes, one of which will be an R.
reniformis GFP gene, can
be efficiently expressed using a single promoterlenhancer to transcribe a
single message. Any
heterologous open reading frame can be linked to IRES elements.. In the
present context, this
means any selected protein that one desires to express and any second reporter
gene (or
selectable marker gene). In this way, the expression of multiple proteins
could be achieved, for
example, with concurrent monitoring through GFP production.
A vector useful according to the invention may also comprise a selectable
marker
allowing identification of a cell that has received a functional copy of the
GFP-encoding gene
CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
construct. In its simplest form, the GFP sequence itself, linked to a chosen
promoter may be
considered a selectable marker, in that illumination of cells or cell lysates
with the proper
wavelength of light and measurement of emitted fluorescence at the expected
wavelength allows
detection of cells that express the GFP construct. In other forms, the
selectable marker may
comprise an antibiotic resistance gene, such as the neomycin, bleomycin,
zeocin or phleomycin
resistance genes, or it may comprise a gene whose product complements a defect
in a host cell,
such ~as the gene t~ncoding dihydrofolate reductase (DHFR), or, for example,
iri yeast, the Leu2
gene. Alternatively, the selectable marker may, in some cases be a luciferase
gene or a
chromogenic substrate-converting enzyme gene such as the ~i-galactosidase
gene.
GFP-encoding sequences according to the invention may be expressed either as
free-
standing polypeptides or frequently as fusions with other polypeptides. It is
assumed that one of
skill in the art can, given the polynucleotide sequences disclosed herein
(e.g., SEQ ID NO: 1)
readily construct a gene comprising a sequence encoding R. reniformis GFP or a
fluorescent
variant thereof and a sequence comprising one or more polypeptides or
polypeptide domains of
interest. It is understood that the fusion of GFP coding sequences and
sequences encoding a
polypeptide of interest maintains the reading frame of all polypeptide
sequences involved. As
used herein, the term "polypeptide of interest" or "domain of interest" refers
to any polypeptide
or polypeptide domain one wishes to fuse to a GFP molecule of the invention.
The fusion of a
GFP polypeptide of the invention with a polypeptide of interest may be through
linkage of the
GFP sequence to either the N or C terminus of the fusion partner, or the GFP
sequence may even
be inserted in frame between the N and C termini of the polypeptide of
interest, if so desired.
Fusions comprising GFP polypeptides of the invention need not comprise only a
singel
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polypeptide or domain in addition of the GFP. Rather, any number of domains of
interest may
be linked in any way as long as the GFP coding region retains its reading
frame and the encoded
polypeptide retains fluorescence activity under at least one set of
conditions. One non-limiting
example of such conditions includes physiological salt concentration (i.e.,
aboutr 90 mM), pH
near neutral and 37°C.
a. Plasmid vectors.
Any plasmid vector that allows expression of a GFP coding sequence of the
invention in
a selected host cell type is acceptable for use according to the invention. A
plasmid vector useful
in the invention may have any or all of the above-noted characteristics of
vectors useful
according to the invention. Plasmid vectors useful according to the invention
include, but are not
limited to the following examples: Bacterial - pQE70, pQE60, pQE-9 (Qiagen)
pBs,
phagescript, psiX174, pBluescript SK, pBsKS, pNHBa, pNHl6a, pNHl8a, pNH46a
(Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRITS (Pharmacia);
Eukaryotic -
pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL
(Pharmacia). However, any other plasmid or vector may be used as long as it is
replicable and
viable in the host.
b. Bacteriophage vectors.
There are a number of well known bacteriophage-derived vectors useful
according to the
invention. Foremost among these are the lambda-based vectors, such as Lambda
Zap II or
Lambda-Zap Express vectors (Stratagene) that allow inducible expression of the
polypeptide
encoded by the insert. Others include filamentous bacteriophage such as the
MI3-based family
of vectors.
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c. Viral vectors.
A number of different viral vectors are useful according to the invention, and
any viral
vector that permits the introduction and expression of sequences encoding R.
reriiformis GFP or
variants thereof in cells is acceptable for use in the methods of the
invention. Viral vectors that
can be used to deliver foreign nucleic acid into cells include but are not
limited to retroviral
vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral
vectors, and Semliki
forest viial (alphaviral) vectors. Defective retroviruses are well
characterized fir use in gene
transfer (for a review see Miller, A.D. (1990) Blood 76:271). Protocols for
producing
recombinant retroviruses and for infecting cells i~a vitro or ira vivo with
such viruses can be found
in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene
Publishing
Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals.
Details of
retrovirus production and host cell transduction of use in the methods of the
invention are also
presented in Example 1, below.
In addition to retroviral vectors, Adenovirus can be manipulated such that it
encodes and
expresses a gene product of interest but is inactivated in terms of its
ability to replicate in a
normal lytic viral life cycle (see for example Berkner et al., 1988,
BioTechniques 6:616;
Rosenfeld et al., 1991, Science 252:431-434; and Rosenfeld et al., 1992, Cell
68:143-155).
Suitable adenoviral vectors derived from the adenovirus strain~Ad type 5 d1324
of other strains of
adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the
art.
Adeno-associated virus (AAV) is a naturally occurring defective virus that
requires another
virus, such as an adenovirus or a herpes virus, as a helper virus for
efficient replication and a
productive life cycle. (For a review see Muzyczka et al., 1992, Curr. Topics
in Micro. and
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Immunol: 158:97-129). An AAV vector such as that described in Traschin et al.
(1985, Mol.
Cell. Biol. 5:3251-3260) can be used to introduce nucleic acid into cells. A
variety of nucleic
acids have been introduced into different' cell types using AAV vectors (see,
for example,
Hermonat et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6466-6470; and Traschin
et al., 1985,
Mol. Cell. Biol. 4: 2072-2081).
Finally, the introduction and expression of foreign genes is often desired in
insect cells
because high level expression may be obtained, the culture conditions are
simple relative to
mammalian cell culture, and the post-translational modifications made by
insect cells closely
resemble those made by mammalian cells. For the introduction of foreign DNA to
insect cells,
such as' Drosophila S2 cells, infection with baculovirus vectors is widely
used. Other insect
vector systems include, for example, the expression plasmid pIZ/VS-His
(InVitrogen) and other
variants of the pIZ/VS vectors encoding other tags and selectable markers.
Insect cells are
readily transfectable using lipofection reagents, and there are lipid-based
transfection products
specifically optimized for the transfection of insect cells (for example, from
PanVera).
2. Host Cells Useful According to the Invention.
Any cell into which a recombinant vector carrying an R. reniformis GFP or
variant
thereof may be introduced and wherein the vector is permitted to drive the
expression of the GFP
or GFP variant sequence is useful according to the invention. That is, because
of the wide
variety of uses for the GFP molecules of the invention, any cell in which a
GFP molecule of the
invention may be expressed and preferably detected is a suitable host. Vectors
suitable for the
introduction of GFP-encoding sequences to host cells from a variety of
different organisms, both
prokaryotic and eukaryotic, axe described herein above or known to those
skilled in the art.
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WO 01/64843 PCT/USO1/06131
Host cells may be prokaryotic, such as any of a number of bacterial strains,
or may be
eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or
mammalian cells
including, for example, rodent, simian or human cells. Cells expressing GFPs
of the invention
may be primary cultured cells, for example, primary human fibroblasts or
keratinocytes, or may
be an established cell line, such as NIH3T3, 293T or CHO cells. Further,
mammalian cells
useful for expression of GFPs of the invention may be phenotypically normal or
oncogenically
transformed. It is assumed that one skilled in the art can readily establish
and maintain a chosen
host cell type in culture.
3. Introduction of GFP-Encoding Vectors to Host Cells.
GFP-encoding vectors may be introduced to selected host cells by any of a
number of
suitable methods known to those skilled in the art. For example, GFP
constructs may be
introduced to appropriate bacterial cells by infection, in the case of E. coli
bacteriophage vector
particles such as lambda or M13, or by any of a number of transformation
methods for plasmid
vectors or for bacteriophage DNA. For example, standard calcium-chloride-
mediated bacterial
transformation is still commonly used to introduce naked DNA to bacteria
(Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, NY), but electroporation may also be used (Ausubel et al. ,
1989; 'supra)..
For the introduction of GFP-encoding constntcts to yeast or other fungal
cells, chemical
transformation methods. are generally used (e.g. as described by Rose et al.,
1990, Methods in
Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
For
transformation of S. cerevisiae, for example, the cells are treated with
lithium acetate to achieve
transformation efficiencies of approximately 104 colony-forming units
(transformed cells)/~,g of
CA 02401544 2002-08-27
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DNA. Transformed cells are then isolated on selective media appropriate to the
selectable
marker used. Alternatively, or in addition, plates or filters lifted from
plates may be scanned for
GFP fluorescence to identify transformed clones.
For the introduction of R. reniformis GFP-encoding vectors to mammalian cells,
the
method used will depend upon the form of the vector. For plasmid vectors, DNA
encoding R.
reniformis GFP or variants thereof may be introduced by any of a number of
transfection
methods, including, for example, lipid-mediated transfection (" lipofection"
), ~DEAE-dextran-
mediated transfection, electroporation or calcium phosphate precipitation.
These methods are
detailed, for example, in Ausubel ~et al., 1989, supra.
Lipofection reagents and methods suitable for transient transfection of a wide
variety of
transformed and non-transformed or primary cells are widely available, making
lipofection an
attractive method of introducing constructs to eulcaryotic, and particularly
mammalian cells in
culture. For example, LipofectAMINETM (Life Technologies) or
LipoTaxiT~''(Stratagene) kits are
available. Other companies offering reagents and methods for lipofection
include Bio-Rad
Laboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBI
Fermentas,
PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals
USA.
For the introduction of R. reniformis GFP-encoding vectors to insect cells,
.such as
Drosophila Schneider 2 cells (S2) cells, Sf~ or Sf~lcells, transfection is
also performed by
lipofection.
Following transfection with an R. reniformis GFP-encoding vector of the
invention,
eukaryotic (preferably, but not necessarily mammalian) cells successfully
incorporating the
construct (infra- or extrachromosomally) may be selected, as noted above, by
either treatment of
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CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
the transfected population with a selection agent, such as an antibiotic whose
resistance gene is
encoded by the vector, or by direct screening using, for example, FRCS of the
cell population or
fluorescence scanning of adherent cultures. Frequently, both types of
screening may be used,
wherein a negative selection is used to enrich for cells taking up the
construct and FACS or
fluorescence scanning is used to further enrich for cells expressing GFPs or
to identify specific
clones of cells, respectively. For example, a negative selection with the
neomycin analog 6418
(Life Technologies, Inc.) may be used to identify cells that have received the
vector, and
fluorescence scanning may be used to identify those cells or clones of cells
that express the R.
reniformis GFP or GFP variant to the greatest extent.
4. Preparation of Antibodies Reactive with R. reniformis GFP
Antibodies that bind to a GFP polypeptide encoded by a polynucleotide of the
invention
are useful, for example, in protein purification and in protein association
assays. An antibody
useful in the invention may comprise a whole antibody, an antibody fragment, a
polyfunctional
antibody aggregate, or in general a substance comprising one or more specific
binding sites from
an antibody. The antibody fragment may be a fragment such as an Fv, Fab or
F(ab')Z fragment or
a derivative thereof, such as a single chain Fv fragment. The antibody or
antibody fragment may
be non-recombinant, recombinant or humanized. The antibody may be of an
immunoglobulin
isotype, e.g., IgG, IgM, and so forth. In addition, an aggregate, polymer,
derivative and
conjugate of an immunoglobulin or a fragment thereof can be used where
appropriate.
GFP-derived peptides used to induce specific antibodies preferably have an
amino acid
sequence consisting of at least five amino acids and more conveniently at
least ten amino acids.
It is advantageous for such peptides to be identical to a region of the
natural R. reniformis GFP
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WO 01/64843 PCT/USO1/06131
protein or variant thereof, and they may even contain the entire amino acid
sequence of R.
reniformis GFP (e.g., SEQ ID NO: 2) or a variant thereof.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, etc.,
may be immunized by injection with peptides or polypeptides having sequences
derived from the
GFP polypeptides of the invention. Depending on the host species, various
adjuvants may be
used to increase the immunological response. Such adjuvants include but are
not limited to
Freund's, mineral .gels such as aluminum hydroxide, and surface active
substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin,
and dinitrophenol.
To generate polyclonal antibodies, the antigen (i.e., an R. reniformis GFP
polypeptide,
variant thereof, or peptide fragment derived therefrom) may be conjugated to a
conventional
Garner in order to increase its immunogenicity, and an antiserum to the
peptide-carrier conjugate
raised. Short stretches of amino acids corresponding to a GFP polypeptide of
the invention may
be fused, either by expression as a fusion product or by chemical linkage,
with amino acids from.
another protein such as keyhole limpet hemocyanin or GST, with antibodies then
being raised
against the chimeric molecule. Coupling of a peptide to a carrier protein and
immunizations may
be performed as described in Dymecki et al., 1992, J. Biol. Chem., 267:4815.
The serum can be
titered against polypeptide antigen by ELISA or alternatively by dot, or spot
blotting (Boersma &
Van Leeuwen, 1994, J. Neurosci. Methods, 51:317). A useful serum will react
strongly with the
appropriate peptides by ELISA, for example, following the procedures of Green
et al., 1982,
Cell, 28:477.
Techniques for preparing monoclonal antibodies are well known, and monoclonal
antibodies may be prepared using an antigen, preferably bound to a carrier, as
described by
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WO 01/64843 PCT/USO1/06131
Arnheiter et al., 1981, Nature, 294:278. Monoclonal antibodies are typically
obtained from
hybridoma tissue cultures or from ascites fluid obtained from animals into
which the hybridoma
tissue was introduced. Monoclonal antibody-producing hybridomas (or polyclonal
sera) can be
screened for antibody binding to the target protein according to methods known
in the art.
5. Variants of R. reniformis GFP According to the Invention.
The invention provides methods of identifying variant R. reniformis GFPs that
are even
better suited, for example, for use in methods employing FRET or for FAGS
analysis than the
wild-type R. reniformis GFP of amino acid sequence SEQ ID NO: 2, encoded by
the
polynucleotide of SEQ ID NO: 1. The wild-type GFP isolated directly from R.
renifonnis
organisms has 3-6-fold higher quantum yield than A. victoria GFP. As shown
herein in Example
4, the R. reniformis GFP polypeptide produced in mammalian cells from.
recombinant nucleic
acid sequences of the invention has spectral characteristics nearly
indistinguishable from the
native polypeptide, i.e., the recombinant R. reniformis GFP of the invention
is 3-6 fold brighter
than that of A. victoria wild-type GFP expressed in the same cell type and has
excitation and
emission spectra similar to the natural R. reniformis GFP protein. However,
even with the
improved brightness of the recombinantly produced R. reniformis GFP over A.
victoria GFP, the
identification of R. reniformis GFP variants with enhanced brightness is
desirable.
In addition to R. reniformis GFP variants with increased brightness, other
modifications
are also of interest. For example, variants exhibiting shifts in either
excitation or emission
spectra or both axe useful since they allow the monitoring of the location or
level of more than
one polypeptide in the same cell through simple fluorescence measurements.
Also, GFP variants
with, for example, an excitation spectrum that is overlapped by the emission
spectrum of another
GFP (wild-type or variant) can be useful for FRET-based assays. Alternatively,
GFP variants
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WO 01/64843 PCT/USO1/06131
whose spectral characteristics are responsive to environmental changes, such
as pH or
oxidation/reduction status or are responsive to changes in phosphorylation
status are useful in
studies of such intracellular or even extracellular changes.
a. Mutagenesis Methods Useful According to the Invention
Modifications to the R. reniformis GFP coding sequences may be either random
or
targeted. In either case, selection involves monitoring individual clones for
the desired modified
characteristic, be it enhanced fluorescence relative to wild-type R.
reniformis~ GFP, a spectral
shift, or other modification.
Many random and site-directed mutagenesis methods are known in the art, and
any of
them that generate modifications to the R. reniformis GFP coding sequence of
SEQ ID NO: 1 are
applicable to generate variant GFPs of the invention. Several examples of both
random and site-
directed mutagenesis are described below.
Random Mutagenesis
Chemical mutagenesis using, for example, nitrous acid, permanganate or formic
acid may
be used to generate random mutations essentially as described by Meyer et al.,
1985, Science
229: 242, which is incorporated herein in its entirety by reference. When
following the Meyer et
al. method, a mutated population of single-stranded R. reniformis GFP
fragments is generated
that is then amplified using the PCR primers used herein 'above for
amplification of wild-type R.
reniformis GFP. The amplification products, bearing random mutations, are
cloned into an
appropriate vector and transformed into bacteria, and colonies are screened
for altered
fluorescence characteristics relative to wild-type R. reniformis GFP either
expressed from the
same vector in the same bacterial strain or purified.
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An alternative to chemical mutagenesis for the generation of random mutants is
the use of
a mutagenic bacterial strain, such as the XL1-Red E. coli strain (Stratagene),
which is deficient
in DNA polymerise proofreading activity .and DNA repair machinery. A plasmid
introduced to
this or a similar strain of bacteria becomes mutated during cell division:
When using a
mutagenic bacterial strain such as XL1-Red, plasmids containing the GFP
sequence to be
mutagenized (i.e., SEQ ID NO: 1) are transformed into the mutagenic bacteria
and propagated
for about two days (shorter or longer, depending upon the desired degree of
mutagenesis). The
randomly mutated plasmids are isolated from the culture using standard methods
and re-
transformed into non-mutagenic bacteria (e.g., E. coli strain DHS ; Life
Technologies, Inc.),
which are plated to achieve individual colonies. The colonies are then
screened for the desired
altered fluorescence characteristic relative to colonies expressing wild-type
R. reniformis from
the same plasmid in the same bacterial strain.
Another example of a method for random mutagenesis is the so-called "error-
prone PCR
method" . As the name implies, the method amplifies a given sequence under
conditions in
which the DNA polymerise does not support high fidelity incorporation. The
conditions
encouraging error-prone incorporation for different DNA polymerises vary,
however one skilled
in the art may determine such conditions for a given enzyme. A key variable
for many DNA
polymerises in the f delity of amplification is, for example, the type and
concentration of
divalent metal ion in the buffer. The use of manganese ion and/or variation of
the magnesium or
manganese ion concentration may therefore be applied to influence the error
rate of the
polymerise. As with the other methods, mutagenized sequences are inserted into
an appropriate
vector, transformed into bacteria and screened for the desired
characteristics.
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Site-Directed or Targeted Mutagenesis
There are a number of site-directed mutagenesis methods known in the art which
allow
one to mutate a particular site or region in a straightforward manner. These
methods are
embodied in a number of kits available commercially for the performance of
site-directed
mutagenesis, including both conventional and PCR-based methods. Examples
include the
EXSITETM PCR-based site-directed mutageriesis kit available from Stratagene
(Catalog No.
200502; PCR based) and the QUII~CHANGETM site-directed mutageriesis kit from
Stratagene
(Catalog No. 200518; PCR based), and the CHAMELEON' double-stranded site-
directed
mutagenesis kit, also from Stratagene (Catalog No. 200509).
Older methods of site-directed mutagenesis known in the art relied upon sub-
cloning of
the sequence to be mutated into a vector, such as an M13 bacteriophage vector,
that allows the
isolation of single-stranded DNA template. In these methods one annealed a
mutagenic primer
(i:e., a primer capable of annealing to the site to be mutated but bearing one
or more mismatched
nucleotides at the site to be mutated) to the single-stranded template and
then polymerized the
complement of the template starting from the 3' end of the mutagenic primer.
The resulting
duplexes were then transformed into host bacteria and plaques were screened
for the desired
mutation.
More recently, site-directed mutagenesis has employed PCR methodologies, which
have
the advantage of not requiring a single-stranded template. In addition,
methods have been
developed that do not require sub-cloning. Several issues must be considered
when PCR-based
site-directed mutagenesis is performed. First, in these methods it is
desirable to reduce the
number of PCR cycles to prevent expansion of undesired mutations introduced by
the
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CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
polymerise. Second, a selection must be employed in order to reduce the number
of non-
mutated parental molecules persisting in the reaction. Third, an extended-
length PCR method is
preferred in order to allow the use of a single PCR primer set. And fourth,
because of the non-
template-dependent terminal extension activity of some thermostable
polymerises it is often
necessary to incorporate an end-polishing step into the procedure prior to
blunt-end ligation of
the PCR-generated mutant product.
The protocol described below accommodates these considerations through the
following
steps. First, the template concentration used is approximately 1000-fold
higher than that used in
conventional PCR reactions, allowing a reduction in the number of cycles from
25-30 down to 5-
without dramatically reducing product yield. Second, the restriction
endonuclease DpnI
(recognition target sequence: 5-Gm6ATC-3, where the A residue is methylated)
is used to select
against parental DNA, since most common strains of E. coli Dam methylate their
DNA at the
sequence 5'-GATC-3'. Third, Taq ~ Extender is used in the PCR mix in order to
increase the
proportion of long (i.e., full plasmid length) PCR products. Finally, Pfu DNA
polymerise is
used to polish the ends of the PCR product prior to intramolecular ligation
using T4 DNA ligase.
The method is described in detail as follows:
PCR-based Site Directed Mutagenesis
Plasmid template DNA (approximately 0.5 pmole) is added to a PCR cocktail
containing:
lx mutagenesis buffer (20 mM Tris HCI, pH 7.5; 8 mM MgClz; 40 ug/m1 BSA); 12-
20 pmole of
each primer (one of skill in the art may design a mutagenic primer as
necessary, giving
consideration to those factors such as base composition, primer length and
intended buffer salt
concentrations that affect the annealing characteristics of oligonucleotide
primers; one primer
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must contain the desired mutation, and one (the same or the other) must
contain a 5' phosphate to
facilitate later ligation), 250 uM each dNTP, 2.5 U Taq DNA polymerase, and
2.5 U of Taq
Extender (Available from Stratagene; See Nielson et al. (1994) Strategies 7:
27, and U.S. Patent
No. 5,556,772). The PCR cycling is performed as follows: I cycle of 4 min at
94°C, 2 min at
50°C and 2 min at 72°C; followed by 5-10 cycles of 1 min at
94°C, 2- min at 54°C and 1 min at
72°C. The parental template DNA and the linear, PCR-generated DNA
incorporating the
mutagenic primer are treated with DpnI (10 U) and Pfu DNA polymerase (2.5U).
This results in
the Dpnl digestion of the in vivo methylated parental template and hybrid DNA
and the removal,
by Pfu DNA polymerase, of the non-template-directed Taq DNA polymerase-
extended bases)
on the linear PCR product. The reaction is incubated at 37°C for 30 min
and then transferred to
72°C for an additional 30 min. Mutagenesis buffer (115 u1 of lx)
containing 0.5 mM ATP is
added to the DpnI-digested, Pfu DNA polymerase-polished PCR products. The
solution is
mixed and I O u1 are removed to a new microfuge tube and T4 DNA ligase (2-4 U)
is added. The
ligation is incubated for greater than 60 min at 37°C. Finally, the
treated solution is transformed
into competent E. coli according to standard methods.
Limited Random Mutagenesis
A subcategory of site-directed mutagenesis involves the use ~ of randomized
oligonucleotides to introduce random mutations into a limited region of a
given sequence (this
will be referred to as "limited random mutagenesis"). This is particularly
useful when one
wishes to mutate every base within, for example, a region encoding a
hexapeptide. Generally,
the oligonucleotides used for this type of approach have a stretch of constant
nucleotides exactly
complementary to a region on either side of and immediately adjacent to the
region to be
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WO 01/64843 PCT/USO1/06131
mutated, linked by a randomized or partially randomized oligonucleotide
sequence
corresponding to the sequence to be mutated. One of the constant sequences
flanking the
mutagenic region should have a restriction site to facilitate the replacement
of wild-type
sequence with the mutagenized sequence following mutagenesis. Ideally, such a
restriction site
is naturally present adj acent to the region to be mutated, but one skilled in
the art may also
introduce restriction sites through silent mutations, without altering the
coding sequence (see, for
example, the list of restriction sites that may be introduced by silent
mutagenesis in the New
England Biolabs (NEB) catalog appendices, specifically at pages 282-283 of the
1998/1999 NEB
catalog).
In the limited random mutagenesis method, mutagenic oligonucleotides as
described
above are used, along with a selected partner primer, and a wild type, or even
previously
mutated, recombinant R. reniformis GFP construct template (wild-type, or,
alternatively;
previously altered) to PCR amplify a pool of fragments, all randomly or semi-
randomly mutated
at the desired sites. The partner primer is selected so that it is either 5'
or 3' of the mutagenized
stretch of nucleotides, and should have either a naturally occurnng
restriction site or an
engineered restriction site that does not alter GFP coding sequences, to
permit the replacement of
the wild-type with the mutated sequences. Conveniently, the partner primer may
bind in the
vector sequences immediately 5' or 3' of the GFP coding sequence. The
amplified pool of
mutated fragments is cleaved with the restriction enzymes recognizing the
respective sites in the
mutagenic and partner primers, and the pool is ligated into a similarly
cleaved recombinant
vector comprising the GFP coding sequences (either 5' of or 3' of the
mutagenized site) not
amplified during the mutagenic step, to generate a pool of full length GFP
coding sequences
randomly or semi-randomly mutated only over the selected stretch of
nucleotides.
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The mutations in the limited random mutagenesis approach are referred to as
"random or
semi-random" because the mutagenic sequences do not necessarily have to be
completely
random. One of skill in the art will recognize, for example, that it is
possible to vary one, two, or
all three nucleotides in a codon with different results as far as the range.of
possible changes to
the peptide sequence encoded, from no change (often possible ~in the third or
"wobble"
nucleotide) to limited change (changes affecting the middle and or third
nucleotide only) to
completely random change (changes affecting all three macleotides of the
codon). Therefore, by
maintaining some nucleotides constant within the mutagenized region and
allowing others to
vary (either over all four possible nucleotides or over one or more subsets of
them), the
characteristics of the mutagenized region may be controlled. Sequences
mutagenized in such a
manner would be "semi-randomly" mutagenized. Following the cloning of the
mutated pool of
R. reniformis GFP vectors using the limited random mutagenesis method, or its
equivalent, the
mutated pool is transformed into bacteria, expression is induced, and the
clones are screened for
the desired altered characteristic.
b. Purification of R. reniformis GFP or Variants Thereof.
If necessary, R. reniformis GFP is purified from R. reniformis organisms as
described by
Ward and Cormier (1979, J. Biol. Chem. 254: 781-788) and by Matthews et al.
(1977,
Biochemistry 16: 85-91), the contents of both of which are herein incorporated
by reference.
Similar procedures may be applied by one of skill in the art to bacterially
expressed R.
reniformis GFP or variants thereof following freeze-thaw lysis and preparation
of a clarified
lysate by centrifugation at 14,000 x g. Briefly, the methods employed by
Matthews et al. and
Ward and Cormier involve successive chromatography over DEAF-cellulose,
Sephadex G-100,
and DTNB (5, 5'-dithiobis(2-nitrobenzoic acid))-Sepharose columns, and
dialysis against 1 mM
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Tris (pH 8.0), 0.1 mM , EDTA. The dialyzed fractions containing GFP
(identified by
fluorescence) are then acid treated to precipitate contaminants, followed by
neutralization of the
supernatant, which is lyophilized. Low salt (10 mM to 1 mM initially) and pH
ranging from 7.5
to 8.5 are critical to~ maintaining activity upon lyophilization. The
lyophilized sample is re-
suspended in water, immediately centrifuged to remove less-soluble
contaminants and applied to
a Sephadex G-75 column. GFP is eluted in 1.0 mM Tris (pH 8.0), 0.1 mM EDTA.
Samples are
concentrated by . partial lyophilization and dialyzed against 5 mM sodium -
acetate, 5,. mM
imidazole, 1 mM EDTA, pH 7.5, followed by chromatography over a DEAE-BioGel-A
column
equilibrated in the same dialysis buffer. GFP is eluted with a continuous
acidic gradient from pH
6.0 to 4.9 in the same acetate/imidizole buffer. Following dialysis of GFP-
containing fractions
against 1.0 mM Tris-HCI, 0.1 mM EDTA, pH 8.0, the sample is partially
lyophilized to
concentrate and passed over a Sephadex G-75 (Superfine) column. The GFP-
containing
fractions are then loaded onto a DEAE-BioGel A column in Tris/EDTA buffer at
pH 8.0,
followed by elution in a continuous alkaline gradient from pH 8.5 to 10.5
formed with 20 mM
glycine, 5 mM Tris-HCl and 5 mM EDTA. GFP-containing fractions contain
essentially
homogeneous R. reniformis GFP.
In screening applications requiring less pure GFP preparations, , recombinant
R.
reniformis or variants thereof can be purified from bacteria as follows.
Bacteria transformed
with a recombinant GFP-encoding vector of the invention are grown in Luria-
Bertani medium
containing the appropriate selective antibiotic (e.g., ampicillin at 50
~,g/ml). If the vector
permits, recombinant polypeptide expression is induced by the addition of the
appropriate
inducer (e.g., IPTG at 1 mM). Bacteria are harvested by centrifugation and
lysed by freeze-thaw
of the cell pellet. Debris is removed by centrifugation at 14,000 x g, and the
supernatant is
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loaded onto a Sephadex G-75 (Pharmacia, Piscataway, NJ) column equilibrated
with 10 mM
phosphate buffered saline, pH 7Ø Fractions containing GFP are identified by
fluorescence
emission at 506 nm when excited by 500 nm light, or by excitation and emission
over a range of
spectra when purifying GFP variants with altered spectral characteristics.
c. Modifications to R. reniformis GFP Useful According to the Invention.
The R. reniformis chromophoric center is comprised of amino acids 64-69 of the
wild-
type polypeptide, which has the sequence FQYGNR. Mutation of this amino acid
sequence at
one or more positions, using for example, standard site-directed or limited
random mutagenesis
or its equivalent, can give rise to R. reniformis variants exhibiting enhanced
fluorescence
intensity or shifted spectral characteristics. Changes at sites outside of the
chromophoric center
may also be affect the fluorescence properties of the polypeptide. For
example, because R.
reniformis lives at a temperature significantly below 37°C, mutations
that stabilize the folded
fluorescent form of the polypeptide at 37°C may enhance the
fluorescence of the polypeptide in
human or mammalian cell culture, or in bacterial cultures, for that matter.
Further, while the
chemical nature of the R. reniformis GFP chromophore is nearly identical to
that of the A.
victoria GFP chromophore (Ward et al., 1980, Photochem. Photobiol. 31: 611-
615), the
fluorescence characteristics, including intensity and spectra are quite
different. This indicates
that modifications outside of the chromophoric center will likely have an
impact on fluorescence
characteristics.
In addition to modifications that change the coding sequence of wild-type R.
reniformis
GFP, the nucleic acid sequence encoding the polypeptide may be modified to
enhance its
expression in mammalian or human cells. The codon usage of R. reniformis is
optimal for
expression in R. reniformis, but not for expression in mammalian or human
systems. Therefore,
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the adaptation of the sequence isolated from the sea pansy for expression in
higher eukaryotes
involves the modification of specific codons to change those less favored in
mammalian or
human systems to those more commonly used in these systems. This so-called
"humanization"
is accomplished by site-directed mutagenesis of the less favored codons as
described herein or as
known in the art. Similar modifications of the A. victoria GFP coding
sequences are described in
U.S. Patent No. 5,874,304. The preferred codons for human gene expression arr
listed in Table
1. The .codons in the table are arranged from left to right in descending
order'of relative use in
human genes. Consideration of the codons in R. reniformis GFP (SEQ ID NO: 1 )
relative to
those favored in human genes allows one of skill in the art to identify which
codons to modify in
the R. reniformis GFP gene to achieve more efficient expression in human or
mammalian cells.
In particular, those codons underlined in the table are almost never used in
known human genes
and, if found in the R. reniformis sequence would therefore represent the most
important codons
to modify for enhanced expression efficiency in mammalian or human cells.
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TABLE 1
PREFERRED DNA CODONS FOR HUMAN USE
Amino Acids Codons Preferred in Human Genes
Alanine Ala A GCC GCT GCA GCG
Cysteine Cys C TGC TGT
Aspartic Asp D GAC GAT
acid
Glutamic Glu E GAG GAA
acid
PhenylalaninePhe F TTC TTT
Glycine Gly G GGC GGG GGA GGT
Histidine His H CAC CAT
Isoleucine Ile I ATC ATT ATA
Lysine Lys K AAG AAA
Leucine Leu L CTG TTG CTT CTA TTA
Methionine Met M ATG
Asparagine Asn N AAC AAT
Proline Pro P CCC CCT CCA CCG
Glutamine Gln Q CAG CAA
Arginine Arg R CGC AGG CGG AGA CGA CGT
Serine Ser S AGC TCC TCT AGT TCA TCG
Threonine Thr T ACC ACA ACT ACG
Valine Val V GTG GTC GTT GTA
Tryprophan Trp W TGG
Tyrosine Tyr Y TAC TAT
The codons at the left represent those most preferred for use in human genes,
with human
usage decreasing towards the right. Underlined codons are almost never used in
human genes.
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6. Screening For R. reniformis GFP Mutants With Altered Fluorescence
Characteristics
or Altered Traits.
One method of screening for altered fluorescence characteristics involves
lifting single
bacterial colonies transformed with a mutated GFP sequence from a plate onto a
support, such as
0.45 ~,m pore size nitrocellulose membranes (Schleicher & Schuell, Keene, NH),
placing the
membranes onto fresh agar/medium plates (e:g., LB agar containing 50 ~g/ml
ampicillin, 1 mM
IPTG for a vector containing ampr and lacI repressor genes, and a lac operator
upstream of the R.
reniformis GFP coding region), bacteria-side up, and allowing colonies to grow
on the
membrane. The membranes are then scanned for fluorescence characteristics of
the colonies.
Scanning may be performed under illumination with monochromatice light, for
example as
generated by passing light from a 150 W Xenon lamp (Xenon Corp., Woburn, MA)
through
interference filters appropriate for the desired excitation wavelengths'
(filters available, for
example, from CVI Laser Corp., Albuquerque, NM). Emissions from the
illuminated colonies
may be observed through, for example, a Schott KV500 filter, which has a 500
nm wavelength
cutoff. The same methods of screening mutants for altered fluorescence
characteristics are
applicable regardless of whether mutagenesis is random or targeted.
Alternative fluorescence scanning equipment includes a scanning polychromatic
light
source (such as a fast monochromator from T.LL.L. Photonics, Munich, Germany)
and an
integrating RGB color camera (such as the Photonic Science Color Cool View).
Following
mufti-wavelength excitation scanning, images captured by the integrating color
camera may be
subjected to image analysis to determine the actual color of the emitted light
using software such
as Spec R4 (Signal Analytics Corp., Vienna, VA, USA).
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With many of the altered characteristics (e.g., fluorescence intensity,
thermal stability or
spectral characteristics) being screened for, bacteria or eukaryotic (e.g.,
yeast or mammalian)
cells expressing the mutated form may first be screened relative to control
cells expressing the
wild-type form, followed if necessary by characterization of either clarified
lysates' or purified
polypeptides from those colonies selected by the cellular screen. For other
altered characteristics
(e.g., pH sensitivity or phosphorylation-dependent alteration of
fluorescence), purified
polypeptides or at least clarified bacterial or eukaryotic cell lysates may be
necessary for
screening. Where necessary, clarified lysate preparation and/or purification
is/are achieved
according to methods described herein or known in the art. Ultimately,
purified mutated or
altered GFP polypeptides can be compared to wild-type R. reniformis GFP
(native or
recombinant) with regard to the characteristic one desires to modify. When
screening for
mutants of R. reniformis GFP with altered fluorescence intensity or brightness
according to- the
invention, one looks.for fluorescence that is at least two times more intense
or bright than the
fluorescence of wild-type R. reniformis GFP (either isolated from R.
reniformis or expressed
from a recombinant vector construct of the invention), and up to 3 times, 5
times, 10 times, 20
times, 50 times or even 100 or more times as intense or bright as the same
molar amount of wild-
type R. renifirmis GFP.
When screening for R. reniformis GFP mutants with altered spectral
characteristics, one
looks for GFP polypeptides that exhibit excitation or emission spectra that
are distinguishable or
detectably distinct from those of the wild-type GFP polypeptide. By
distinguishable or
detectably distinct is meant that standard filter sets allow either the
excitation of one form
without excitation of the other form, or similarly, that standard filter sets
allow the distinction of
the emission from one form from the other. Generally, distinguishable
excitation or emission
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spectra have peaks that vary by more than 1 nm, and preferably vary by more
than 2, 3, 4, 5, 10
or more nm. The peaks of distinguishable spectra are also preferably narrow,
covering a range of
about 5 nm or less, 7 nm or less, 10 nm or less, 15 nm or less, 20 nm or less,
50 nm or less, or
100 nm or less. The maximum allowable breadth of a peak that is considered
distinguishable is
directly related to how much the peak maximum varies from the maximum of the
peak it is being
distinguished from. In other words, the larger the variance between the peak
wavelengths of two
fluorescent polypeptides, the broader the peaks may be and still be
distinguishable. Conversely,
the lower the variance between the centers of the peaks, the narrower the
peaks must be to be
distinguishable.
Particularly preferred spectral shifts are shifts in emission spectra that are
not
accompanied by distinguishable shifts in excitation spectra. Such a shift
permits the excitation
of two or more different GFPs with light of the same wavelength (or same range
of excitation
wavelengths) yet also permits distinction of the fluorescence of two or more
GFPs based on the
different emission wavelengths.
Other preferred spectral shifts include those that render the R. reniformis
GFP capable of
FRET as either a donor or an acceptor fluoroprotein. For example, a spectral
alteration that
changes the excitation spectrum of a first fluorescent polypeptide so that it
overlaps the emission
spectrum of a second fluorescent polypeptide will define a pair of fluorescent
polypeptides
capable of FRET. It is preferred, although not necessary that both the first
and second
fluorescent polypeptides be GFP polypeptides; if a non-GFP fluorescent
polypeptide is a donor
or acceptor for FRET, it is preferred that a polynucleotide sequence for that
fluorescent
polypeptide is known.
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If both fluorescent polypeptides of a FRET pair are R. reniformis GFP
polypeptides, one
or both polypeptides may be altered. That is, one may be wild-type R.
reniformis GFP and the
other may be altered, or both GFPs of the FRET pair may be altered. In the
case in which wild-
type R. reniformis GFP is a member of the pair, it may be either the donor or
the acceptor
member of the pair.
Another altered characteristic that may enhance the usefulness of the R.
reniformis GFf
polypeptides of the invention is altered stability of the polypeptide in vivo.
As mentioned above,
modifications that alter the folded stability of the polypeptide's fluorophore
center can alter the
fluorescence intensity of the polypeptide. However, modifications that
increase or reduce the in
vivo or in vitro half life of the entire GFP polypeptide, i.e., modifications
that affect polypeptide
turnover or degradation are also useful. For example, increased stability can
enhance the
detection of the modified R. reniformis GFP by allowing a larger steady-state
pool of GFP. to
accumulate at a given expression rate. Importantly, there is also usefulness
for R. reniformis
GFP polypeptide variants with rechaced in vivo or in vitro stability. For
example, the
responsiveness of reporter assays for transcription is enhanced by reporter
molecules with shorter
half lives. Generally, the shorter the biological half life of the reporter
molecule, the faster a
new steady state is achieved when the transcription rate increases or
decreases, enhancing the
sensitivity of the assay.
II. How to Use R. reniformis GFP and Variants Thereof According to the
Invention.
R. reniformis GFP and variants thereof according to the invention are useful
in a number
of different ways. Generally, R. reniformis is useful in any process or assay
that can be
performed with A. victoria GFP. Further, because of its superior spectral
characteristics and
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fluorescent intensity, wild-type R. reniformis GFP is useful in processes and
assays beyond those
that can be performed with A. victoria GFP. And finally, altered, modified or
mutated R.
reniformis is even more useful for particular applications of fluorescent
marker technologies.
R. reniformis GFP or variants thereof may be used as selectable .markers for
the
identification of cells transfected or infected with a gene transfer vector.
In this aspect, cells
transfected with a construct encoding GFP may be identified over a background
of non-
transfected or infected cells by illumination of the cells with light within
the excitation spectrum
and detection of fluorescent emission in the emission spectrum of the GFP.
The usefulness of R. reniformis GFP as a reporter molecule stems from
properties such as
ready detection, the feasibility of real-time detection in vivo, and the fact
that the introduction of
a substrate is not required. R. reniformis gfp genes can therefore be used to
identify transformed
cells (e.g., by fluorescence-activated cell sorting (FACS) or fluorescence
microscopy),. to
measure gene expression in vitro and in vivo, to label specific cells in
multicellular organisms
(e.g., to study cell lineages), to label and locate fusion proteins, and to
study intracellular protein
trafficking. Variant R. reniformis GFPs exhibiting altered fluorescence
characteristics in
response to changes in, for example, pH, phosphorylation status or redox
status are useful for
studying changes in those parameters in vivo.
R. reniformis.GFPs may also be used for standard biological applications. For
example,
they may be used as molecular weight markers on protein gels and Western
blots, in calibration
of fluorometers and FACS equipment and as a marker for micro inj ection into
cells and tissues.
In methods to produce fluorescent molecular weight markers, an R. reniformis
GFP gene
sequence is fused to one or more DNA sequences that encode proteins having
defined amino
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sequences, and the fusion proteins are expressed from an expression vector.
Expression results
in the production of fluorescent proteins of defined molecular weight or
weights that may be
used as markers.
Preferably, purified fluorescent proteins are subjected to size-fractionation,
such as by
using a gel. A determination of the molecular weight of an unknown protein is
then made by
compiling a calibration curve from the fluorescent standards and reading the
unknown molecular
weight from the curve.
A. Uses of R. reniformis GFPs With Altered Emission Spectra.
Amino acid replacements in R. reniformis GFP that produce different color
emission
spectra permit simultaneous use of multiple reporter genes. Different colored
R. reniformis GFPs
can be used to identify multiple cell populations in a mixed cell culture or
to track multiple cell
types, permitting differences in cell movement or migration to be visualized
in real time without
the need to add additional agents or fix or kill the cells:
Other options involving the uses of GFPs with altered emission spectra include
tracking
and determining the ultimate location of multiple proteins within a single
cell, tissue or
organism. Differential promoter analysis in which gene expression from two
different promoters
is determined in the same cell, tissue or organism is also permitted by GFPs
with differing
emission spectra, as is and FAGS sorting of mixed cell populations.
In tracking proteins within a cell, the R. reniformis GFP variants are used in
a manner
analogous to fluorescein and rhodamine to tag interacting proteins or subunits
whose
association is then be monitored dynamically in intact cells by FRET. Cells
are irradiated with
light at the excitation wavelengths of the donor, and emission by the acceptor
is monitored to
indicate protein: protein interactions of tagged proteins.
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The techniques that can be used with spectrally separable R. reniformis GFP
derivatives
are exemplified by confocal microscopy, flow cytometry, and fluorescence
activated cell sorting
(FACS) using modular flow, dual excitation techniques.
B. Use of R. reniformis GFP in the Identification of Transfected Cells::
R. reniformis GFP may be introduced as a selectable marker to identify
transfected cells
from a background of non-transfected cells. Alternatively, R. reniformis GFP
transfection may
be used to pre-label isolated cells or a population of similar cells prior to
exposing the cells to an
environment in which different cell types are present. Detection of GFP in
only the original cells
allows the location of such cells to be determined and compared with the total
population.
Cells that have been transfected with exogenous DNA can be identified with the
R.
reniformis GFPs of the invention. out creating a fusion protein. The method
relies on the
identification of cells that have received a plasmid or vector that comprises
,at least ,two
transcriptional or translational units. A first unit will encode and direct
expression of the desired
protein, while the second unit will encode and direct expression of R.
reniformis GFP or a
variant thereof. Co-expression of GFP from the second transcriptional or
translational unit
ensures that cells containing the vector are detected and differentiated from
cells that do not
contain the vector.
The R. reniformis GFP sequences ofthe.inventiori rnay also be fused~to a DNA
sequence encoding a selected protein in order to directly label the encoded
protein with GFP.
Expressing such an R. reniformis GFP fusion protein in a cell results in the
production of
fluorescently-tagged proteins that can be readily detected. This is useful in
confirming
that a protein is being produced by a chosen host cell. It also allows the
location of the selected
protein to be determined, whether this represents a natural location or
whether the protein has
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been artificially targeted to another location.
C. Analysis of Transcriptional Regulatory Sequences.
The R. reniformis GFP genes of the invention allow a range of transcriptional
regulatory
sequences to be tested for their suitability for use with a given
gene, cell, or system. This applies to in vitro uses, such as in identifying a
suitable transcriptional
regulatory sequence for use in recombinant expression and high level protein
production, as well
as in vivo uses, such as in pre-clinical testing or in gene therapy in human
subjecas.
In order to analyze a transcriptional regulatory sequence, one must first
establish a
control cell or system. In the control, a positive result is established by
using a known and
effective promoter, such as the CMV promoter. To test a candidate
transcriptional regulatory
sequence, another cell or system is established in which all conditions are
the same except for
there being different transcriptional regulatory sequences in the expression
vector or, genetic
construct.
After running the assay for the same period of time and under the same
conditions as in
the control, the GFP expression levels are determined. This allows one to make
a comparison of
the strength or suitability of the candidate transcriptional regulatory
sequence with the standard
or control transcriptional regulatory sequence.
Transcriptional regulatory sequences that can be tested in this manner also
include
candidate tissue-specific promoters and candidate-inducible promoters. Testing
of tissue-specific
promoters allows the identification of optimal transcriptional regulatory
sequences for use with a
given cell. Again, this is useful both in vitro and in vivo. Optimizing the
combination of a given
transcriptional regulatory sequence and a given cell type in recombinant
expression and protein
production is often necessary to ensure that the highest possible expression
levels are achieved.
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The GFP encoded by a regulatory sequence testing construct may optionally have
a
secretion signal fused to it, such that GFP secreted to the medium is
detected.
The use of tissue-specific promoters and inducible promoters is particularly
powerful in
vivo embodiments. When used in the context of expressing a therapeutic gene in
an animal, the
use of such transcriptional regulatory sequences allows expression only in a
given tissue or
tissues, at a given site and/or under defined conditions. Achieving tissue-
specific expression is
particularly important in certain gene therapy applications, such as in the -
expression of a
cytotoxic agent, as is often employed in approaches to the treatment of
cancer. In expressing
other therapeutic genes with a beneficial effect, rather than a cytotoxic
effect, tissue-specific
expression is also . preferred since it can optimize the effect of the
treatment. Appropriate
tissue-specific and inducible transcriptional regulatory sequences are known
to those of skill in
the art, or, for example, described herein above.
D. Use of R. reniformis GFP in Assays for Compounds That Modulate
Transcription.
R. reniformis GFP and variant: thereof are useful in screening assays to
detect
compounds that modulate transcription. In this aspect of the invention, R.
reniformis GFP coding
sequences are positioned downstream of a promoter that is known to be
inducible by the agent
that one wishes to detect. Expression of GFP in the cells will normally be
silent, and is activated
by exposing the cell to a composition that contains the selected agent. In
using a promoter that is
responsive to, for example, a lipid soluble transcriptional modulator, a
toxin, a hormone, a
cytokine, a growth factor or other defined molecule, the presence the
particular defined molecule
can be determined. For example, an estrogen-responsive regulatory sequence may
be linked to
GFP in order to test for the presence of estrogen in a sample.
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It will be clear to one of skill in the art that any of the detection assays
may be used in the
context of screening for agents that inhibit, suppress or otherwise down
regulate gene expression
from a given transcriptional regulatory sequence. Such negative effects are
detectable by
decreased GFP fluorescence that results when gene expression is down-regulated
in response to
the presence of an inhibitory agent.
E. Use of R. reniformis GFP and Variants Thereof in FACS Analyses.
Many conventional FACS methods require the use of fluorescent dyes conjugated
to
purified antibodies. Fusion proteins tagged with a fluorescent label are
preferred over antibodies
in FAGS applications because the cells do not have to be incubated with the
fluorescent-tagged
reagent and because there is no background due to nonspecific binding of an
antibody conjugate.
GFP is particularly suitable for use in FACS as fluorescence is stable and
species-independent
and does not require any substrates or cofactors.
As with other expression embodiments, a desired protein may be directly
labeled with
..GFP by preparing and expressing a GFP fusion protein in a cell. GFP can also
be co-expressed
from a second transcriptional or translational unit within the expression
vector that expresses
desired protein, as described above. Cells expressing the GFP-tagged protein
or cells
co-expressing GFP are then detected and sorted by FACS analysis. An advantage
of GFP from
R. reniformis is that its excitation and emission spectra are amenable to
standard optics and filter
sets used in FACS analyses.
F. Other Uses of R. reniformis GFP Fusion Proteins.
R: reniformis GFP genes can be used as one portion of a fusion protein,
allowing the
location of the tagged protein to be identified. Fusions of GFP with an
exogenous protein should
CA 02401544 2002-08-27
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preserve both the fluorescence of GFP and functions of the host protein, such
as physiological
functions and/or targeting functions.
Both the amino and carboxyl termini of GFP may be fused to virtually any
desired
protein to create an identifiable GFP-fusion, and fusion may be mediated by a
linker sequence if
necessary to preserve the function of the fusion partner.
R. reniformis GFP fusions are useful for subcellular localization studies.
Localization
studies have previously been carried out by subcellular fractionation and by
immunofluorescence. However, these techniques can give only a static
representation of the
position of the protein at one instant in the cell cycle. In addition,
artifacts can be introduced
when cells are fixed for immunofluorescence. Using GFP to visualize proteins
in living cells,
which allows proteins to be followed throughout the cell cycle in an
individual cell, is thus an
important technique.
R. reniformis GFP can be used to analyze intracellular protein traffic in
mammalian and
human cells under a variety of conditions in real time. Artifacts resulting
from fixing cells are
avoided. In these applications, R. reniformis GFP is fused to a known protein
in order to
examine its sub-cellular location under different natural conditions.
EXAMPLES
Example 1. Production of Infectious R. reniformis GFP Retroviruses.
Virus production was carried out by co-transfecting 293T cells with 3 ~,g each
of the
vectors pGPhisD (Stratagene), pVSV-G-puro (Stratagene), and either pFB-rGFP or
the vector
pFB-AvGFP. The latter vector contains a copy of the A. victoria GFP gene that
includes an
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insertion of the alanine codon GCT immediately following the methionine
initiation codon to
accommodate the inclusion of-a Kozak consensus sequence, as well as the Ser-
>Thr "red shift"
amino acid substition at position 65 (relative to the wt sequence). The
vectors pGPhisD and
pVSV-G-puro encode the viral proteins gag-pol and VSV-G, which are required in
t~ahs for
production of virus.
The transfections were carned out using the MBS Transfection Kit (Stratagene),
with
some modifications. For each transfection, 2.5x106 293T cells were plated iri
a 60 mm tissue
culture dish. The following day medium was aspirated and replaced with 4 ml
pre-warmed
DMEM supplemented with 7% MBS and 25 qM chloroquine (Sigma, St. Louis, MO)
prior to
transfection. The DNA/CaP04 transfection mixes were prepared according to the
manufacturer's
recommended protocol and added to the cells. After a 3 h incubation, the
medium was replaced
with 4 ml of pre-warmed complete culture medium (DMEM containing 10% Fetal
Bovine Serum
(FBS)) supplemented with 25 ~.M chloroquine and incubated for 6-7 hours. The
medium was
then replaced with 4 ml of pre-warmed DMEM + 10% FBS. Cells .were incubated
overnight
(12-16 hours), and medium was replaced with 3 ml pre-warmed DMEM + 10% FBS,
and virus
was collected overnight (24 hours). The 3 ml viral supernatant was removed and
filtered through
a .45 qm filter. Supernatants were stored on ice for immediate use or frozen
on dry ice and
stored at -80 C.
Example 2. Transduction of Host Cells with R. reniformis GFP Retroviral
Stocks.
One day prior to transduction, NIH3T3 cells were plated in DMEM supplemented
with
10% Calf Serum (CS) at 1 x 105 cells/well in a 6 well tissue culture dish. The
following day the
viral supernatants were serially diluted in DMEM + 10% CS to a final volume of
1.0 ml/sample,
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and supplemented with DEAF-Dextran (Sigma, St. Louis, MO, catalog #D-9885) to
a final
concentration of 10 p,g/ml. Culture medium was removed from the NIH3T3 cells
and replaced
with 1 ml of viral dilution. Each diluted viral sample was applied to a well
containing the
NIH3T3 cells, and incubated for 3 h, after which 1 ml of pre-warmed DMEM + 10%
CS was
added to each well, and the plates were then incubated for 2 d. After 2 d the
plates were washed
2x with PBS, trypsinized, pelleted by centrifugation, and resuspended in 1.0
ml PBS. ~ Cell
suspensions were stored on ice and analyzed by Fluorescence Activated Cell
'Sorting (FRCS)
within one hour. FAGS analysis was performed by Cytometry Research Services,
(Sorrento
Valley, CA).
Example 3. Transfection of CHO Cells and Extract Preparation.
CHO cells were transfected with the plasmid pFB-rGFP using Lipofectaminer
(BRL)
according to the manufacturers recommendations. Two days following
transfection, soluble
protein extracts were prepared from transfected and untransfected CHO cells by
first washing the
cells 2x with PBS, and then subjecting the cells to three freeze-thaw cycles
in 0.25 M Tris-HCI,
pH 7.8. The lysates were cleared by high speed centrifugation, and the
supernatants were then
used for spectral analyses.
Example 4. Spectral Analysis of Recombinant R, reniformis GFP.
Excitation and emission spectral analysis was determined using a Shimadzu RF-
1501
Spectrofluorophotometer. Excitation and emission scans were performed on equal
amounts of
total protein prepared from transfected or untransfected CHO cells. Background
fluorescence
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was subtracted from the scans of the GFP-containing (transfected) extract by
normalization to
the scans of the untransfected extracts.
In order to compare the fluorescence profile for the cloned R. reriifo~~2is
protein to that
for the purified native protein, excitation and emission scans were carried
out using soluble
protein extracts from CHO cells transfected with the expression vector. As
shown in Figure 4,
the fluorescence profile for the cloned protein is virtually identical to that
reported for the native
protein, with a single major excitation peak at 500 nm (compared with 498 nm
for the native
protein) preceded by a vibrational shoulder at approximately 470 nm, a
characteristic of the
native RecZilla GFPs. The emission spectra show a single peak at 506 nm for
the cloned protein,
compared with the reported maximum of 509 nm for the native protein.
Example 5. Preparation of a Humanized R. reniformis GFP Polynucleotide.
Expression of ectopic genes in.the cells of a particular species is very often
enhanced if
the polynucleotide sequence of the gene is altered to make use of codons that
are preferred in
highly expressed genes endogenous to the cell type of choice. For example, the
"humanization"
of the red-shifted Aeqzcof~ea GFP resulted in a dramatic enhancement of the
level of fluorescence
when expressed in mammalian cells (Yang, T.-T. et. al [1996] Nucl. Acids Res.
24[22]:4592-
4593).
The inventors have altered 166 of the gene's 238 codons such that all of the
codons in the
resulting gene are biased for high expression in human cells. The codon
changes were based
upon the human codon usage preferences described in Haas et al., 1996, Curr
Biol. 6[3]: 315-
4593. The codon usage preferences shown in Table 1 are equivalent to those in
the Haas
reference.
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Cell culture. 293, 293T and CHO cells were maintained at 37 °C at 5%
C02 in Dulbecco's
Modifed Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (Gemini Bio-
Products,
Inc.) and 1 % glutamine.
Construction of the hrGFP gene. The humanized recombinant GFP (hrGFP)
nucleotide sequence
was altered according to Haas, J. et. al., 1996, Cacrr. Biol. 6[3]:315-324,
such that all the codons
were selected based on their prevalence in genes that are highly expressed in
human cells. The
sequence is set forth in SEQ ID NO: 3 (see Figure 5). Figure 6 shows a
sequence alignment of
the non-humanized recombinant R. reniformis GFP (SEQ ID NO: 1) and humanized
R.
reniformis GFP polynucleotide sequences. The humanized gene was constructed by
synthesizing
a set of complementary, overlapping oligonucleotides which were annealed,
ligated and
subcloned. Both strands were completely sequenced, and mutations were
corrected using the
QuickChange kit (Stratagene). The PCR fragment was digested to completion with
EcoR I and
Xlzo I and inserted between the EcoR I and Xho I sites of the retroviral
expression vector pFB
(Stratagene) to create the vector pFB-hrGFP. This vector was used for further
analysis of the
humanized gene.
Virus production. Virus production was carried out by co-transfecting 293T
cells with 3 ~g each
of the vectors pGPhisD (Stratagene), pVSV-G-puro (Stratagene), and either pFB-
hrGFP or the
vector pFB-EGFP. The latter vector contains a copy of the fully humanized,
redshifted A.
victoria GFP gene (EGFP). The vectors pGPhisD and pVSV-G-puro encode the viral
proteins
gag-pol and VSV-G, which are required in t~°ahs for production of
virus. The transfections were
carried out using the MBS Transfection Kit (Stratagene), with some
modifications. For each
transfection, 2.5x106 293T cells were plated in a 60 mm tissue culture dish.
The following day
CA 02401544 2002-08-27
WO 01/64843 PCT/USO1/06131
medium was aspirated and replaced with 4 ml pre-warmed DMEM supplemented with
7% MBS
and 25 ~,M chloroquine (Sigma, St. Louis, MO) prior to transfection. The
DNA/CaPO4
transfection mixes were prepared according to the manufacture's recommended
protocol and
added to the cells. After a 3 h incubation, the medium was replaced with 4 ml
of pre-warmed
complete culture medium (DMEM containing 10% FBS) supplemented with 25 ~M
chloroquine
and incubated for 6-7 hours. The medium was then replaced with 4 ml pre-warmed
DMEM +
10% FBS. Cells were incubated overnight (12-16 hours), and medium was replaced
with 3 ml
pre-warmed DMEM + 10% FBS, and virus was collected overnight (24 hours). The 3
ml viral
supernatant was removed and filtered through a .45 pin filter. Supernatants
were stored on ice
for immediate use or frozen on dry ice and stored at -80°C.
Example 6. Evaluation of the expression of R. reniformis GFP from a humanized
polynucleotide
sequence.
The humanized R. reniformis GFP coding sequence described in Example 5 has
been
tested for expression in several human, rodent and monkey cell lines.
Fluoresence levels have
been found to be substantially higher for the humanized rGFP (hrGFP) gene
compared with that
for rGFP. In a direct comparison between cell populations harboring single
copy proviral
expression cassettes encoding either hrGFP or the humanized, red-shifted
Aeqicot~ea GFP
(EGFP), we found relative fluorescence intensity to be comparable between the
two genes.
Viral Transduction. One day prior to transduction, 293 cells (human) or CHO
cells (hamster)
were plated in DMEM supplemented with 10% FBS at 1 x 105 cells/well in a 6
well tissue
culture dish. The following day the viral supernatants were serially diluted
in DMEM + 10%
FBS to a final volume of 1.0 ,ml/sample, and supplemented with DEAF-Dextran
(Sigma, St.
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WO 01/64843 PCT/USO1/06131
Louis, MO, catalog #D-9885) to a final concentration of 10 ~.g/ml. Culture
medium was removed
from the target cells and replaced with 1 ml of viral dilution. Each diluted
viral sample was
applied to a well containing the target cells, and incubated for 3 h, after
which 1 ml of pre-
warmed DMEM + 10% FBS was added to each well, and the plates were then
incubated for 2 d.
After 2 d the plates were washed 2x with PBS, trypsinized, pelleted by
centrifugation, and
resuspended in,1.0 ml PBS. Cell suspensions were stored on ice and analyzed by
Fluorescence
Activated Cell Sorting (FACS) within one hour. FAGS analysis was;performed by
Cytometry
Research Services, (Sorrento Valley, CA).
Comparison of rGFP and hrGFP expression in vivo. To determine whether the
sequence
alterations introduced into the R. ~eyaiformis GFP gene resulted in enhanced
expression, the
hrGFP coding sequence was inserted into the vector pFB, and the resulting
vector pFB-hrGFP
was transfected side-by-side with the parental vector pFB-rGFP gene into CHO
cells. Visual
inspection of the transfected cells by fluorescence microscopy (excitation 450-
490 nm; emission
520 nm) revealed a dramatic enhancement of fluorescence for the hrGFP gene
compared with
rGFP (data not shown). CHO cells were next infected with virus derived from
the two vectors at
equivalent multiplicities of infection (MOI), and two days following infection
the transduced
cells were analyzed by fluorescence-activated cell sorting (FACS; excitation
488 nm, .emission
515-545 nm). As the results in Figure 7. indicate, the majority of the cell
population transduced
with pFB-hrGFP fluoresces approximately 2-3 orders of magnitude brighter than
cells harboring
pFB-rGFP.
The relative fluorescence was compared from cells harboring single-copy
proviral
integrants encoding rGFP, hrGFP or EGFP. 293 cells were infected at low MOI,
and two days
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WO 01/64843 PCT/USO1/06131
post-infection the fluoresence levels were analysed by FACS. As shown in
Figure 8,
supernatants that were diluted to 1:1000 or greater resulted in target
populations in which
approximately 10% or less of the cells were transduced; in such populations
the.vast majority of
the cells are expected to have single copy proviral integrants. In the
transduced populations, the
overall fluorescence intensity of the populations were comparable for the
hrGFP and EGFP
expression vectors. Fluorescence for rGFP was significantly lower than for the
latter two genes.
Similar results were obtained for experiments involving the transduction of
HeLa, CHO, COS7
and NIH3T3 cells (data not shown).
OTHER EMBODIMENTS
Other embodiments will be evident to those of skill in the art. It should be
understood
that the foregoing detailed description is provided for clarity only and is
merely exemplary.
The spirit and scope of the present invention are not limited to the above
examples, but are
encompassed by the following claims.
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