U.S. patent application number 10/457982 was filed with the patent office on 2003-11-13 for fluorescent protein sensors for measuring the ph of a biological sample.
Invention is credited to Llopis, Juan, Remington, S. James, Tsien, Roger Y., Wachter, Rebekka M..
Application Number | 20030212265 10/457982 |
Document ID | / |
Family ID | 22244686 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030212265 |
Kind Code |
A1 |
Tsien, Roger Y. ; et
al. |
November 13, 2003 |
Fluorescent protein sensors for measuring the pH of a biological
sample
Abstract
Disclosed are fluorescent protein sensors for measuring the pH
of a sample, nucleic acids encoding them, and methods of use. The
preferred fluorescent protein sensors are variants of the green
fluorescent protein (GFP) from Aequora victoria. Also disclosed are
compositions and methods for measuring the pH of a specific region
of a cell, such as the mitochondrial matrix or the Golgi lumen.
Inventors: |
Tsien, Roger Y.; (La Jolla,
CA) ; Llopis, Juan; (San Diego, CA) ; Wachter,
Rebekka M.; (Creswell, OR) ; Remington, S. James;
(Eugene, OR) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
22244686 |
Appl. No.: |
10/457982 |
Filed: |
June 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10457982 |
Jun 9, 2003 |
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09602641 |
Jun 22, 2000 |
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6608189 |
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09602641 |
Jun 22, 2000 |
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09094359 |
Jun 9, 1998 |
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6140132 |
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Current U.S.
Class: |
536/23.2 ;
435/320.1; 435/325; 435/4; 435/455; 435/69.1; 530/350 |
Current CPC
Class: |
G01N 2333/43595
20130101; C07K 14/43595 20130101; Y10S 435/81 20130101; G01N 33/84
20130101 |
Class at
Publication: |
536/23.2 ;
530/350; 435/69.1; 435/320.1; 435/325; 435/4; 435/455 |
International
Class: |
C12Q 001/00; C07H
021/04; C07K 014/435; C12P 021/02; C12N 005/06; C12N 015/85 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. NS27177, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A polynucleotide comprising a nucleotide sequence encoding a
functional engineered fluorescent protein whose amino acid sequence
is substantially identical to the amino acid sequence of Aequorea
green fluorescent protein (SEQ ID NO:2) and whose emission
intensity changes as pH varies between 5 and 10.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the protein includes the substitutions S65G/S72A/T203Y/H231L in
the amino acid sequence of Aequorea green fluorescent protein (SEQ
ID NO:2).
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the protein includes the substitutions
S65G/V68L/Q69K/S72A/T203Y/H231L in the amino acid sequence of
Aequorea green fluorescent protein (SEQ ID NO:2).
4. The polynucleotide of claim 1, wherein the amino aid sequence of
the protein includes the substitutions
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/- N164H/H231L in the amino
acid sequence of Aequorea green fluorescent protein (SEQ ID
NO:2).
5. The polynucleotide of claim 1, wherein the amino acid sequence
of the protein includes the substitution H148G in the amino acid
sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
6. The polynucleotide of claim 1, wherein the amino acid sequence
of the protein includes the substitution H148Q in the amino acid of
Aequorea green fluorescent protein (SEQ ID NO:2).
7. An expression vector comprising expression control sequences
operatively linked to a polynucleotide molecule comprising a
nucleotide sequence encoding a functional engineered fluorescent
protein whose amino acid sequence is substantially identical to the
amino acid sequence of Aequorea green fluorescent protein (SEQ ID
NO:2) and whose emission intensity changes as pH varies between 5
and 10.
8. A recombinant host cell comprising the expression vector of
claim 7.
9. The recombinant host cell of claim 8, wherein the recombinant
host cell is a prokaryotic cell.
10. The recombinant host cell of claim 8, wherein the recombinant
host cell is a eukaryotic cell.
11. A method of determining the pH of a region of a cell
comprising: introducing into the cell a polynucleotide encoding a
polypeptide including an indicator having a first fluorescent
protein moiety whose emission intensity changes as pH varies
between 5 and 10; culturing the cell under conditions that permit
expression of the polynucleotide; exciting the indicator; and
determining the intensity of the light emitted by the first protein
moiety at a first wavelength, wherein the emission intensity of the
first fluorescent protein moiety indicates the pH of the region of
the cell in which the indicator is present.
12. The method of claim 11, wherein the polypeptide encoded by the
polynucleotide further includes a targeting sequence linked by a
peptide bond to the indicator.
13. The method of claim 11, wherein the amino acid sequence of the
first fluorescent protein moiety includes the substitutions
S65G/S72A/T203Y/H231L in the amino acid sequence of Aequorea green
fluorescent protein (SEQ ID NO:2).
14. The method of claim 11, wherein the amino acid sequence of the
first fluorescent protein moiety includes the substitutions
S65G/V68L/Q69K/S72A/T203Y/H231L in the amino acid sequence of
Aequorea green fluorescent protein (SEQ ID NO:2).
15. The method of claim 11, wherein the amino acid sequence of the
first fluorescent protein moiety includes the substitutions
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in the amino acid
sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
16. The method of claim 11, wherein the amino acid sequence of the
first fluorescent protein moiety includes the substitution H148G in
the amino acid sequence of Aequorea green fluorescent protein (SEQ
ID NO:2).
17. The method of claim 11, wherein the amino acid sequence of the
first fluorescent protein moiety includes the substitution H148Q in
the amino acid sequence of Aequorea green fluorescent protein (SEQ
ID NO:2).
18. The method of claim 12, wherein the amino acid of the targeting
sequence comprises the amino terminal 81 amino acids of human type
II membrane-anchored protein galactosyltransferase.
19. The method of claim 12, wherein the targeting sequence
comprises the amino terminal 12 amino acids of the presequence of
subunit IV of cytochrome c oxidase.
20. A kit useful for the detection of the pH in a sample, the kit
comprising carrier means containing one or more containers
comprising a first container containing a polynucleotide comprising
a nucleotide sequence encoding a functional engineered fluorescent
protein whose amino acid sequence is substantially identical to the
amino acid sequence of Aequorea green fluorescent protein (SEQ ID
NO:2) and whose emission intensity changes as pH varies between 5
and 10.
21. The kit of claim 20, wherein the amino acid sequence of the
protein includes the substitutions S65G/S72A/T203Y/H231L in the
amino acid sequence of Aequorea green fluorescent protein (SEQ ID
NO:2).
22. The kit of claim 20, wherein the amino acid sequence of the
protein includes the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L
in the amino acid sequence of Aequorea green fluorescent protein
(SEQ ID NO:2).
23. The kit of claim 20, wherein the amino acid sequence of the
protein includes the substitutions
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H2- 31L in the amino
acid sequence of Aequorea green fluorescent protein (SEQ ID
NO:2).
24. The kit of claim 20, wherein the amino acid sequence of the
protein includes the substitution H148G in the amino acid sequence
of Aequorea green fluorescent protein (SEQ ID NO:2).
25. The kit of claim 20, wherein the amino acid sequence of the
protein includes the substitution H148Q in the amino acid sequence
of Aequorea green fluorescent protein (SEQ ID NO:2).
Description
[0001] This application is a continuation-in-part of currently
pending U.S. Ser. No. 09/094,359 (herein incorporated by
reference), which was filed Jun. 9, 1998.
FIELD OF THE INVENTION
[0003] The invention relates generally to compositions and methods
for measuring the pH of a sample and more particularly to
fluorescent protein sensors for measuring the pH of a biological
sample.
BACKGROUND OF THE INVENTION
[0004] The pH within various cellular compartments is regulated to
provide for the optimal activity of many cellular processes. In the
secretory pathway, posttranslational processing of secretory
proteins, the cleavage of prohormones, and the retrieval of escaped
luminal endoplasmic reticulum proteins are all pH-dependent.
[0005] Several techniques have been described for measuring
intracellular pH. Commonly used synthetic pH indicators can be
localized to the cytosol and nucleus, but not selectively in
organelles other than those in the endocytotic pathway. In
addition, some cells are resistant to loading with cell-permeant
dyes because of physical barriers such as the cell wall in
bacteria, yeast, and plants, or the thickness of a tissue
preparation such as brain slices.
[0006] Several methods have been described for measuring pH in
specific regions of the cell. One technique uses microinjection of
fluorescent indicators enclosed in liposomes. Once inside the cell,
the liposomes fuse with vesicles in the trans-Golgi, and the pH of
the intracellular compartments is determined by observing the
fluorescence of the indicator. This procedure can be laborious, and
the fluorescence of the indicator can be diminished due to leakage
of the fluorescent indicator from the Golgi, or flux of the
fluorescent indicator out of the Golgi as part of the secretory
traffic in the Golgi pathway. In addition, the fusion of the
liposomes and components of the Golgi must take place at 37.degree.
C.; however, this temperature facilitates leakage and flux of the
fluorescent indicator from the Golgi.
[0007] A second method for measuring pH utilizes retrograde
transport of fluorescein-labeled verotoxin 1B, which stains the
entire Golgi complex en route to the endoplasmic reticulum. This
method can be used, however, only in cells bearing the receptor
globotriaosyl ceramide on the plasma membrane, and it may be
limited by the residence time of the verotoxin in transit through
the Golgi.
[0008] In a third method, intracellular pH has been measured using
the chimeric protein CD25-TGN38, which cycles between the
trans-Golgi network and the plasma membrane. At the plasma
membrane, the CD25-motif binds extra-cellular anti-CD25 antibodies
conjugated with a pH-sensitive fluorophore. Measurement of
fluorescence upon return of the bound complex to the Golgi can be
used to measure the pH of the organelle.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery that proteins
derived from the Aequora victoria green fluorescence protein (GFP)
show reversible changes in fluorescence over physiological pH
ranges.
[0010] Accordingly, in one aspect, the invention provides a method
for determining the pH of a sample by contacting the sample with an
indicator including a first fluorescent protein moiety whose
emission intensity changes as the pH varies between 5 and 10,
exciting the indicator, and the determining the intensity at a
first wavelength. The emission intensity of the first fluorescent
protein moiety indicates the pH of the sample.
[0011] In another aspect, the invention provides a method for
determining the pH of a region of a cell by introducing into the
cell a polynucleotide encoding a polypeptide including a first
fluorescent protein moiety whose emission intensity changes as the
pH varies between 5 and 10, culturing the cell under conditions
that permit expression of the polynucleotide, and determining the
intensity at a first wavelength. The emission intensity of the
first fluorescent protein moiety indicates the pH of the
sample.
[0012] In a further aspect, the invention provides a functional
engineered fluorescent protein whose amino acid sequence is
substantially identical to the amino acid sequence of the 238 amino
acid Aequora victoria green fluorescence protein shown in FIG. 3 of
U.S. Ser. No. 08/911,825 (SEQ ID NO:______), and whose emission
intensity changes as pH varies between 5 and 10.
[0013] In another aspect, the invention provides a polynucleotide
encoding the functional engineered fluorescent protein.
[0014] The invention also includes a kit useful for the detection
of pH in a sample, e.g., a region of a cell. The kit includes a
carrier means containing one or more containers comprising a first
container containing a polynucleotide encoding a polypeptide
including a first fluorescent protein moiety whose emission
intensity changes as the pH varies between 5 and 10.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a schematic diagram depicting fluorescent protein
sensors used as indicators of intracellular pH.
[0016] FIGS. 2A and 2B are graphs showing absorbance as a function
of wavelength for the fluorescent protein pH sensor EYFP (SEQ ID
NO:______) at various wavelengths (FIG. 2A), and the pH dependency
of fluorescence of various GFP fluorescent protein sensors in vitro
and in cells (FIG. 2B). The fluorescence intensity of purified
recombinant GFP mutant protein (solid symbols) as a function of pH
was measured in a microplate fluorometer. The fluorescence of the
Golgi region of HeLa cells expressing proteins having the 81
N-terminal amino acids of the type II membrane-anchored protein
galactosyltransferase (GT:UDP-galactose-.beta.,-
1,4-galactosyltransferase. EC 2.4.1.22) ("GT") fused to EYFP, or
EGFP, i.e., GT-EYFP or GT-EGFP (open symbols) was determined during
pH titration with the ionophores monensin/nigericin in high KCL
solutions.
[0017] FIGS. 3A and 3B are graphs showing ratiometric measurements
of pH.sub.G by cotransfecting HeLa cells with polynucleotides
encoding GT-ECFP and GT-EYFP. FIG. 3A is a graph showing single
wavelength fluorescence intensities of GT-EYFP and GT-ECFP in the
Golgi region of a HeLa cell. FIG. 3B is a graph showing the ratio
of GT-EYFP/GT-ECFP fluorescence in the same cell as a function of
time.
[0018] FIG. 4 is a graph showing the change in mitochondrial pH of
HeLa cells expressing YFP H148G.
[0019] FIG. 5 is a graph showing the mitochondrial pH of chick
skeletal myotubes expressing YFP H148G in the mitochondrial
matrix.
[0020] FIG. 6 is a graph showing fluorescence and pH in HeLa cells
expressing YFP H148Q targeted to the mitochondrial matrix.
[0021] FIG. 7 is a graph showing a ratiometric measurement of
mitochondrial pH following expression of YFP H148G (pH sensitive)
and GFP T203I (pH insensitive) in mitochondria of HeLa cells.
[0022] FIG. 8 is a graph showing normalized absorbance of WT GFP
and YFP in 75 mM phosphate pH 8.0, 140 mM NACl. Solid lines, WT
GFP; Dashed lines, YFP.
[0023] FIG. 9 is a stereoview of the 2F.sub.o-F.sub.c electron
density map of the YFP chromophore and the stacked Tyr203 after
refinement. The 2.5 .ANG. resolution map was contoured at +1
standard deviation.
DETAILED DESCRIPTION
[0024] The invention provides genes encoding fluorescent sensor
proteins, or fragments thereof, whose fluorescence is sensitive to
changes in pH at a range between 5 and 10. The proteins of the
invention are useful for measuring the pH of a sample. The sample
can be a biological sample and can include an intracellular region
of a cell, such as the lumen of the mitochondria or golgi. The pH
of a sample is determined by observing the fluorescence of the
fluorescent sensor protein.
[0025] The fluorescent protein pH sensor have a broad applicability
to cells and organisms that are amenable to gene transfer. Problems
associated with the use of other agents used to measure pH, e.g.,
problems associated with permeabilizing cells to ester-containing
agents, leakage of agents, or hydrolysis of agents are avoided.
With the fluorescent protein pH sensors of the invention, no
leakage occurs over the course of a typical measurement, even when
the measurement is made at 37.degree. C.
[0026] Compositions and methods described herein also avoid the
need to express and purify large quantities of soluble recombinant
protein, purify and label it in vitro, microinject it back into
cells. An important advantage of the fluorescent protein pH sensors
of the invention is that they can be delivered to cells in the form
of polynucleotides encoding the protein sensor fused to a targeting
signal or signals. The targeting signal directs the expression of
the protein sensors to restricted cell locations. Thus, it is
possible to measure the pH of a precisely defined cellular region
or organelle.
[0027] Polynucleotides and Polypeptides
[0028] In a first aspect, the invention provides a functional
engineered fluorescent protein whose amino acid sequence is
substantially identical to the 238 amino acid Aequora victoria
green fluorescence protein shown in FIG. 3 of U.S. Ser. No.
08/911,825 (SEQ ID NO:______). The term "fluorescent protein"
refers to any protein capable of emitting light when excited with
appropriate electromagnetic radiation, and which has an amino acid
sequence that is either natural or engineered and is derived from
the amino acid sequence of Aequorea-related fluorescent protein.
The term "fluorescent protein pH sensor" refers to a fluorescent
protein whose emitted light varies with changes in pH from 5 to
10.
[0029] The invention also includes functional polypeptide fragments
of a fluorescent protein pH sensor. As used herein, the term
"functional polypeptide fragment" refers to a polypeptide which
possesses biological function or activity which is identified
through a defined functional assay and which is associated with a
particular biologic, morphologic, or phenotypic alteration in the
cell. The term "functional fragments of a functional engineered
fluorescent protein" refers to fragments of a functional engineered
protein that retain a function of the engineered fluorescent
protein, e.g., the ability to fluoresce in a pH-dependent manner
over the pH range 5 to 10. Biologically functional fragments can
vary in size from a polypeptide fragment as small as an epitope to
a large polypeptide.
[0030] Minor modifications of the functional engineered fluorescent
protein may result in proteins which have substantially equivalent
activity as compared to the unmodified counterpart polypeptide as
described herein. Such modifications may be deliberate, as by
site-directed mutagenesis, or may be spontaneous. All of the
polypeptides produced by these modifications are included herein as
long as the pH-dependent fluorescence of the engineered protein
still exists.
[0031] A functional engineered fluorescent protein includes amino
acid sequences substantially the same as the sequence set forth in
SEQ ID NO:______, and whose emission intensity changes as pH varies
between 5 and 10. In some embodiments the emission intensity of the
functional engineered fluorescent protein changes as pH varies
between 5 and 8.5.
[0032] By "substantially identical" is meant a protein or
polypeptide that retains the activity of a functional engineered
protein, or nucleic acid encoding the same, and which exhibits at
least 80%, preferably 85%, more preferably 90%, and most preferably
95% homology to a reference amino acid or nucleic acid sequence.
For polypeptides, the length of comparison sequences will generally
be at least 16 amino acids, preferably at least 20 amino acids,
more preferably at least 25 amino acids, and most preferably 35
amino acids. For nucleic acids, the length of comparison sequences
will generally be at least 50 nucleotides, preferably at least 60
nucleotides, more preferably at least 75 nucleotides, and most
preferably 110 nucleotides.
[0033] By "substantially identical" is meant an amino acid sequence
which differs only by conservative amino acid substitutions, for
example, substitution of one amino acid for another of the same
class (e.g., valine for glycine, arginine for lysine, etc.) or by
one or more non-conservative substitutions, deletions, or
insertions located at positions of the amino acid sequence which do
not destroy the function of the protein (assayed, e.g., as
described herein). Preferably, such a sequence is at least 85%,
more preferably 90%, more preferably 95%, more preferably 98%, and
most preferably 99% identical at the amino acid level to one of the
sequences of EGFP (SEQ ID NO:______), EYFP (SEQ ID NO: ______),
ECFP (SEQ ID NO: ______), EYFP-V68L/Q69K (SEQ ID NO:______), YFP
H148G (SEQ ID NO:______), or YFP H148Q (SEQ ID NO:______).
[0034] Homology is typically measured using sequence analysis
software (e.g., Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). Such software matches
similar sequences by assigning degrees of homology to various
substitutions, deletions, substitutions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine,
threonine; lysine, arginine; and phenylalanine, tyrosine.
[0035] In some embodiments, the amino acid sequence of the protein
includes one of the following sets of substitutions in the amino
acid sequence of the Aequora green fluorescent protein (SEQ ID
NO:______): F64L/S65T/H231L, referred to herein as EGFP (SEQ ID
NO:______); S65G/S72A/T203Y/H231L, referred to herein as EYFP (SEQ
ID NO:______); S65G/V68L/Q69K/S72A/T203Y/H231L, referred to herein
as EYFP-V68L/Q69K (SEQ ID NO:______);
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L, referred to
herein as ECFP (SEQ ID NO:______). The amino acid sequences of
EGFP, EYFP, ECFP, and EYFP-V68L/Q69K are shown in Tables 1-4,
respectively. The amino acids are numbered with the amino acid
following the initiating methionine assigned the `1` position.
Thus, F64L corresponds to a substitution of leucine for
phenylalanine in the 64th amino acid following the initiating
methionine.
1TABLE 1 EGEP Amino Acid Sequence (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK- FICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYOONTPIGDGPVLLPDNH
YLSTOSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0036]
2TABLE 2 EYFP Amino Acid Sequence (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK- FICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPONEKRDHMVLLEFVTAAGITLGMDELYK
[0037]
3TABLE 3 EYFP-V68L/Q69K Amino Acid Sequence (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG- DATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEG- YVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSH- N
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0038]
4TABLE 4 ECFP Amino Acid sequence (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLK- FICT
TGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHN
VYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0039] In other embodiments, the amino acid sequence of the protein
is based on the sequence of the wild-type Aequora green fluorescent
protein, but includes the substitution H148G (SEQ ID NO:______) or
H148Q (SEQ ID NO:______). In specific embodiments, these
substitutions can be present along with other substitutions, e.g.,
the proteins can include the substitutions
S65G/V68L/S72A/H148G/Q80R/T203Y (SEQ ID NO:______), which is
referred to herein as the "YFP H148G mutant,"
S65G/V68L/S72A/H148Q/Q80- R/T203Y, which is referred to herein as
the "YFP H148Q mutant" (SEQ ID NO:______), the as well as
EYFP-H148G (SEQ ID NO: ______) and EFP-H148Q (SEQ ID NO: ______).
The amino acid sequences of these mutants are shown in Tables 5-8,
respectively.
5TABLE 5 Amino Acid Sequence of YFP H148G (SEQ ID NO:_)
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT- YGKLTLKFICTT
GKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQ- ERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
[0040]
6TABLE 6 Amino Acid Sequence of YFP H148Q (SEQ ID NO:_)
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT- YGKLTLKFICTT
GKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQ- ERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
[0041]
7TABLE 7 Amino Acid Sequence of EYFP-H148G (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG- DATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEG- YVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSG- N
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0042]
8TABLE 8 Amino acid. Sequence of EYFP-H148Q (SEQ ID NO:_)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG- DATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEG- YVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQ- N
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0043] In some embodiments, the protein or polypeptide is
substantially purified. By "substantially pure protein or
polypeptide" is meant an functional engineered fluorescent
polypeptide which has been separated from components which
naturally accompany it. Typically, the protein or polypeptide is
substantially pure when it is at least 60%, by weight, free from
the proteins and naturally-occurring organic molecules with which
it is naturally associated. Preferably, the preparation is at least
75%, more preferably at least 90%, and most preferably at least
99%, by weight, of the protein. A substantially pure protein may be
obtained, for example, by extraction from a natural source (e.g., a
plant cell); by expression of a recombinant nucleic acid encoding a
functional engineered fluorescent protein; or by chemically
synthesizing the protein. Purity can be measured by any appropriate
method, e.g., those described in column chromatography,
polyacrylamide gel electrophoresis, or by HPLC analysis.
[0044] A protein or polypeptide is substantially free of naturally
associated components when it is separated from those contaminants
which accompany it in its natural state. Thus, a protein or
polypeptide which is chemically synthesized or produced in a
cellular system different from the cell from which it naturally
originates will be substantially free from its naturally associated
components. Accordingly, substantially pure polypeptides include
those derived from eukaryotic organisms but synthesized in E. coli
or other prokaryotes.
[0045] The invention also provides polynucleotides encoding the
functional engineered fluorescent protein described herein. These
polynucleotides include DNA, cDNA, and RNA sequences which encode
functional engineered fluorescent proteins. It is understood that
all polynucleotides encoding functional engineered fluorescent
proteins are also included herein, as long as they encode a protein
or polypeptide whose fluorescent emission intensity changes as pH
varies between 5 and 10. Such polynucleotides include naturally
occurring, synthetic, and intentionally manipulated
polynucleotides. For example, the polynucleotide may be subjected
to site-directed mutagenesis. The polynucleotides of the invention
include sequences that are degenerate as a result of the genetic
code. Therefore, all degenerate nucleotide sequences are included
in the invention as long as the amino acid sequence of the
functional engineered fluorescent protein or derivative is
functionally unchanged.
[0046] Specifically disclosed herein is a polynucleotide sequence
encoding a functional engineered fluorescent protein that includes
one of the following sets of substitutions in the amino acid
sequence of the Aequora green fluorescent protein (SEQ ID
NO:______): S65G/S72A/T203Y/H231L, S65G/V68L/Q69K/S72A/T203Y/H231L,
or K26R/F64L/S65T/Y66W/N146I/M153T/V163A- /N164H/H231L. In specific
embodiments, the DNA sequences encoding EGFP, EYFP, ECFP,
EYFP-V68L/Q69K, YFP H148G, and YFP H148Q are those shown in Table
9-16 (SEQ ID NOs: ______ to ______), respectively.
[0047] The nucleic acid encoding functional engineered fluorescent
proteins may be reflect the codon choice in the native A. victoria
coding sequence, or, alternatively, may be chosen to reflect the
optimal codon frequencies used in the organism in which the
proteins will be expressed. Thus, nucleic acids encoding a target
functional engineered protein to be expressed in a human cell may
have use a codon choice that is optimized for mammals, or
especially humans.
9TABLE 9 EGFP Nucleic Acid Sequence
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC (SEQ
ID NO:_) GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGG-
CGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC-
CGTGCCCTGGCCCACC CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCC-
CTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTA-
CGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGA-
GGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTT-
CAAGGAGGACGGCAACATCCTGGGGCAC AAGCTCGAGTACAACTACAACAGCCACAA-
CGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCG-
CCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCC-
CATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC
CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
[0048]
10TABLE 10 EYFP Nucleic Acid Sequence
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC (SEQ
ID NO:_) GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC-
GAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAG-
CTGCCCGTGCCCTGGCCCACC CTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTC-
GCCCGCTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATGCCCGAA-
GGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGC-
GCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATC-
GACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGC-
CACAACGTCTATATCATGGCCGACAAGCAGAAGAAC
GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC
GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC
CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
[0049]
11TABLE 11 ECFP Nucleic Acid Sequence
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC (SEQ
ID NO:_) GGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGC-
GAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAG-
CTGCCCGTGCCCTGGCCCACC CTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTC-
AGCCGCTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATGCCCGAA-
GGCTACGTCCAGGAGCGTACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGC-
GCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATC-
GACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACATCAGC-
CACAACGTCTATATCACCGCCGACAAGCAGAAGAAC
GGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC
GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC
CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
[0050]
12TABLE 12 EYFP-V68L/Q69K Nucleic Acid Sequence
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAG- CTGGAC (SEQ
ID NO:_) GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC-
GAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACC-
GGCAAGCTGCCCGTGCCCTGGCCCACC CTCGTGACCACCTTCGGCTACGGCCTGAAG-
TGCTTCGCCCGCTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATG-
CCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAG-
ACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG
GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC
GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC
GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC
CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
[0051]
13TABLE 13 Nucleotide Sequence of the YFP H148G Coding Region
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTC- CCAATTCTTGTTGAATTAGATGGT (SEQ
ID NO:_)
GATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGA
AAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT
GTCACTACTTTCGGTTATGGTCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGG
CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTC
AAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTT
AATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACAC- AAA
TTGGAATACAACTATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAG- AATGGA
ATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAA- CTAGCAGAC
CATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCA- GACAACCATTAC
CTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGA- GACCACATGGTCCTT
CTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGAT- GAACTATACAAA
[0052]
14TABLE 14 Nucleotide Sequence of the YFP H148Q Coding Region+HZ,32
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTT- GTTGA
ATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG
AAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACT
GGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGG
TCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTT
TCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTC
AAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGA
TACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATG
GAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTA
TACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAAT
TAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAAC
AAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC
CTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCA
CATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGG
ATGAACTATACAAA
[0053]
15TABLE 15 Nucleotide Sequence of the EYFP-H148G Coding Region
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCG- GGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGC- GTGTCCGGCGAGG
GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC- TGCACC
ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA
CGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT
TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC
TTCAAGGACCACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG
CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG
ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCGGCAAC
GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA
GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC
AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA
TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA
TGGACGAGCTGTACAAGTAA
[0054]
16TABLE 16 Nucleotide Sequence of the EYFP-H148Q Coding Region
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCG- GGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGC- GTGTCCGGCGAGG
GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC- TGCACC
ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA
CGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT
TCTTCAAGTCCGCCATGCCCCAAGGCTACGTCCAGGAGCGCACCATCTTC
TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG
CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG
ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAGAAC
GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA
GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC
AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC
TACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA
TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA
TGGACGAGCTGTACAAGTAA
[0055] The term "polynucleotide" refers to a polymeric form of
nucleotides of at least 10 bases in length. The nucleotides can be
ribonucleotides, deoxyribonucleotides, or modified forms of either
type of nucleotide. The term includes single and double stranded
forms of DNA. By "isolated polynucleotide" is meant a
polynucleotide that is not immediately contiguous with both of the
coding sequences with which it is immediately contiguous (one on
the 5' end and one on the 3' end) in the naturally occurring genome
of the organism from which it is derived. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector, e.g., an expression vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote, or which exists as a separate molecule
(e.g., a cDNA) independent of other sequences.
[0056] A "substantially identical",nucleic acid sequence codes for
a substantially identical amino acid sequence as defined above.
[0057] The functional engineered fluorescent protein can also
include a targeting sequence to direct the fluorescent protein to
particular cellular sites by fusion to appropriate organellar
targeting signals or localized host proteins. A polynucleotide
encoding a targeting sequence can be ligated to the 5' terminus of
a polynucleotide encoding the fluorescence such that the targeting
peptide is located at the amino terminal end of the resulting
fusion polynucleotide/polypeptide. The targeting sequence can be,
e.g., a signal peptide. In the case of eukaryotes, the signal
peptide is believed to function to transport the fusion polypeptide
across the endoplasmic reticulum. The secretory protein is then
transported through the Golgi apparatus, into secretory vesicles
and into the extracellular space or, preferably, the external
environment. Signal peptides which can be utilized according to the
invention include pre-pro peptides which contain a proteolytic
enzyme recognition site. Other signal peptides with similar
properties to pro-calcitonin described herein are known to those
skilled in the art, or can be readily ascertained without undue
experimentation.
[0058] The targeting sequence can also be a nuclear localization
sequence, an endoplasmic reticulum localization sequence, a
peroxisome localization sequence, a mitochondrial localization
sequence, or a localized protein. Targeting sequences can be
targeting sequences which are described, for example, in "Protein
Targeting", chapter 35 of Stryer, L., Biochemistry (4th ed.). W. H.
Freeman, 1995. The localization sequence can also be a localized
protein. Some important targeting sequences include those targeting
the nucleus (KKKRK) (SEQ ID NO: ______), mitochondrion (the 12
amino terminal amino acids of the cytochrome c oxidase subunit IV
gene (SEQ ID NO:______), or the amino terminal sequence
MLRTSSLFTRRVQPSLFRNILRLQST (SEQ ID NO:______), endoplasmic
reticulum (KDEL (SEQ ID NO:______) at the C-terminus, assuming a
signal sequence present at N-terminus), peroxisome (SKF at
C-terminus), prenylation or insertion into plasma membrane (CaaX,
CC, CXC, or CCXX at C-terminus), cytoplasmic side of plasma
membrane (fusion to SNAP-25), or the Golgi apparatus (fusion to the
amino terminal 81 amino acids of human type II membrane-anchored
protein galactosyltransferase (SEQ ID NO:______), or fusion to
furin).
[0059] Examples of targeting sequences linked to functional
engineered fluorescent proteins include GT-EYFP (SEQ ID NO:______),
GT-ECFP (SEQ ID NO:______), GT-EGFP (SEQ ID NO:______), and
GT-EYFP-V68L/Q69K (SEQ ID NO:______), which are targeted to the
Golgi apparatus using sequences from the GT protein; and EYFP-mito
(SEQ ID NO:______) and EGFP-mito (SEQ ID NO:______), which are
targeted to the mitochondrial matrix using sequences from the amino
terminal region of the cytochrome c oxidase subunit IV gene. The
EYFP, ECFP, EGFP, and EYFP-V68L/Q69K amino acid sequences, as well
as nucleic acids encoding these polypeptides, are described above.
The GT-derived targeting sequence corresponds to the 81 amino
terminal amino acids of the human GT sequence. The GT amino acid
sequences, and the polynucleotide sequences encoding the GT amino
acid sequences, are described in Genbank Accession No. M70427 and
Mengle-Gaw et al., Biochem. Biophys. Res. Commun. 176 (3),
1269-1276 (1991).
[0060] Amino acid sequences of mito-ECFP, mito-EYFP, GT-EGFP, and
GT-EYFP, mito-YFP H148G, mito-YFP H148Q, mito-EYFP H148G, mito
YFP-H148Q are shown in Tables 17-24.
[0061] In specific embodiments, nucleic acid sequences encoding
targeting sequences linked to functional engineered fluorescent
proteins have the sequences shown in Tables 23-32.
17TABLE 17 mito-ECFP Amino Acid Sequence (SEQ ID NO:_)
MLSLRQSIRFFKRSGIMVSKGEELFTGVVPILVELDGDV- NGHRFSVSGEG
EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHM- KQHDF
FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED
GNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQ
QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYK
[0062]
18TABLE 18 mito-EYFP Amino Acid Sequence (SEQ ID NO:_)
MLSLRQSIRFFKRSGIMVSKGEELFTGVVPILVELDGDV- NGHKFSVSGEG
EGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHM- KQHDF
FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED
GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ
QNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYK
[0063]
19TABLE 19 GT-EGFP Amino Acid Sequence (SEQ ID NO:_)
MRLREPLLSGAAMPGASLQRACRLLVAVCALHLGVTLVYYL- AGRDLSRLP
QLVGVSTPLQGGSNSAAAIGQSSGELRTGGAMDPMVSKGEELFTGVV- PIL
VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
YGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFE
GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKR
DHMVLLEFVTAAGITLGMDELYK
[0064]
20TABLE 20 GT-EYFP Amino Acid Sequence (SEQ ID NO:_)
MRLREPLLSGAAMPGASLQPACRLLVAVCALHLGVTLVYYT- AGRDLSRLP
QLVGVSTPLQGGSNSAAAIGQSSGELRTGGAMDPMVSKGEELFTGVV- PIL
VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFG
YGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFE
GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKR
DHMVLLEFVTAAGITLGMDELYK*
[0065]
21TABLE 21 mito-YFP-H148G Amino Acid Sequence (SEQ ID NO:__)
MLRTSSLFTRRVQPSLFRNILRLQSTSKGEELF- TGVVPILVELDGDVNGH
KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTF- GYGLQCFARYP
DHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTPAEVKFEGDTLVN- RIEL
KGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIEDGS
VQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVT
AAGITHGMDELYK
[0066]
22TABLE 22 mito-EYFP-H148Q Amino Acid Sequence (SEQ ID NO:_)
MLRTSSLFTRRVQPSLFRNILRLQSTSKGEELF- TGVVPILVELDGDVNGH
KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTF- GYGLQCFARYP
DHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN- PJEL
KGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGS
VQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVT
AAGITHGMDELYK
[0067]
23TABLE 23 mito-EYFP-H148G Amino Acid Sequence (SEQ ID NO:_)
MLRTSSLFTRRVQPSLFRNILRLQSTMVSKGEE- LFTGVVPILVELDGDVN
GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT- TFGYGVQCFAR
YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL- VNRI
ELKGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIED
GSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
VTAAGITLGMDELYK
[0068]
24TABLE 24 mito-EYFP-H148Q Amino Acid Sequence (SEQ ID NO:_)
MLRTSSLFTRRVQPSLFRNILRLQSTMVSKGEE- LFTGVVPILVELDGDVN
GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT- TFGYGVQCFAR
YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL- VNRI
ELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIED
GSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
VTAAGITLGMDELYK
[0069]
25TABLE 25 GT-ECFP Nucleic Acid Sequence
ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATCCCAGGCGCGTCCCTACAGCGG (SEQ
ID NO:_) GCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGC-
GTCACCCTCGTTTACTAC CTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTC-
GGAGTCTCCACACCGCTGCAG GGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCC-
TCCGGGGAGCTCCGGACCGGAGGG GCCATGGATCCCATGGTGAGCAAGGGCGAGGAG-
CTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCAC-
AGGTTCAGCGTGTCCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTG-
AAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACC-
CTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCC
GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG
CGTACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG
GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC
ATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGAC
AAGCAGAAGAACGGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGC
GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG- CTG
CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG- AAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC- ATGGACGAG
CTGTACAAGTAA
[0070]
26TABLE 26 mito-EYFP Nucleic Acid Secpence
ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCA- AG (SEQ
ID NO:_) GGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCG-
AGCTGGACGGCGACGTAAAC GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG-
ATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCPAGCTGCCCG-
TGCCCTGGCCCACCCTCGTGACCACC TTCGGCTACGGCGTGCAGTGCTTCGCCCGCT-
ACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACG-
TCCAGGAGCGCACCATCTTCTTCAAGGACGAC GGCAACTACAAGACCCGCGCCGAGG-
TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC
GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC
AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAKGAACGGCATCAAGGTG
AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG
CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTAC
CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC
GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
[0071]
27TABLE 27 GT-EGFP Nucleic Acid Sequence
ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATGCCAGGCGCGTCCCTACAGCGG (SEQ
ID NO:_) GCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGC-
GTCACCCTCGTTTACTAC CTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTC-
GGAGTCTCCACACCGCTGCAG GGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCC-
TCCGGGGAGCTCCGGACCGGAGGG GCCATGGATCCCATGGTGAGCAAGCGCGAGGAG-
CTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCAC-
AAGTTCAGCGTGTCCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTG-
AAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACC-
CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCC
GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG
CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG
GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC
ATCCTGGGGcACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC
AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC
GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG- CTG
CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG- AAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC- ATGGACGAG
CTGTACAAGTAA
[0072]
28TABLE 28 GT-EYFP Nucleic Acid Sequence
ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATGCCAGGCGCGTCCCTACAGCGG (SEQ
ID NO:_) GCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGC-
GTCACCCTCGTTTACTAC CTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTC-
GGAGTCTCCACACCGCTGCAG GGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCC-
TCCGGGGAGCTCCGGACCGGAGGG GCCATGGATCCCATGGTGAGCAAGGGCGAGGAG-
CTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCAC-
AAGTTCAGCGTGTCCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTG-
AAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACC-
TTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCC
GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG
CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG
GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC
ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC
AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC
GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG- CTG
CCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAG- AAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC- ATGGACGAG
CTGTACAAGTAA
[0073]
29TABLE 29 mito-YFP H148G Nucleic Acid Sequence
ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGAGT- AAAGGA (SEQ
ID NO:_) GAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTT-
GAATTAGATGGTGATGTTAATGGG CACAAATTTTCTGTCAGTGGAGAGGGTGAAGGT-
GATGCAACATACGGAAAACTTACCCTT AAATTTATTTGCACTACTGGAAAACTACCT-
GTTCCATGGCCAACACTTGTCACTACTTTC GGTTATGGTCTTCAATGCTTTGCAAGA-
TACCCAGATCATATGAAACGGCATGACTTTTTC AAGAGTGCCATGCCCGAAGGTTAT-
GTTCAGGAAAGAACTATATTTTTCAAAGATGACGGG
AACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAG
TTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC
TATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAAC
TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAA
AATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAA
TCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTT- GTA
ACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA
[0074]
30TABLE 30 mito-YFP H148Q Nucleic Acid Sequence
ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGAGT- AAAGGA (SEQ
ID NO:_) GAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTT-
GAATTAGATGGTGATGTTAATGGG CACAAATTTTCTGTCAGTGGAGAGGGTGAAGGT-
GATGCAACATACGGAAAACTTACCCTT AAATTTATTTGCACTACTGGAAAACTACCT-
GTTCCATGGCCAACACTTGTCACTACTTTC GGTTATGGTCTTCAATGCTTTGCAAGA-
TACCCAGATCATATGAAACGGCATGACTTTTTC AAGAGTGCCATGCCCGAAGGTTAT-
GTTCAGGAAAGAACTATATTTTTCAAAGATGACGGG
AACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAG
TTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC
TATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAAC
TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAA
AATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAA
TCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTT- GTA
ACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA
[0075]
31TABLE 31 mito-EYFP-H148G Nucleic Acid Sequence (SEQ ID NO:_)
ATGCTGAGCCTGCGCCAGAGCATCCGCTTCT- TCAAGCGCAGCGGCATCAT
GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTG- CCCATCCTGGTCG
AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC- GAGGGC
GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC
CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACG
GCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTC
TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT
CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG
ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGAC
GGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCGGCAACGT
CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA
TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG
CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA
CCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATC
ACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG
GACGAGCTGTACAAGTAA
[0076]
32TABLE 32 mito-EYFP-H148Q Nucleic Acid Sequence (SEQ ID NO:_)
ATGCTGAGCCTGCGCCAGAGCATCCGCTTCT- TCAAGCGCAGCGGCATCAT
GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTG- CCCATCCTGGTCG
AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC- GAGGGC
GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC
CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACG
GCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTC
TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT
CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG
ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGAC
GGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAGAACGT
CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA
TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG
CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA
CCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATC
ACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG
GACGAGCTGTACAAGTAA
[0077] The fluorescent indicators can be produced as proteins fused
to other fluorescent indicators or targeting sequences by
recombinant DNA technology. Recombinant production of fluorescent
proteins involves expressing nucleic acids having sequences that
encode the proteins. Nucleic acids encoding fluorescent proteins
can be obtained by methods known in the art. For example, a nucleic
acid encoding the protein can be isolated by polymerase chain
reaction of cDNA from A. victoria using primers based on the DNA
sequence of A. victoria green fluorescent protein. PCR methods are
described in, for example, U.S. Pat. No. 4,683,195; Mullis, et al.
Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987), and Erlich,
ed., PCR Technology, (Stockton Press, NY, 1989). Mutant versions of
fluorescent proteins can be made by site-specific mutagenesis of
other nucleic acids encoding fluorescent proteins, or by random
mutagenesis caused by increasing the error rate of PCR of the
original polynucleotide with 0.1 mM MnCl.sub.2 and unbalanced
nucleotide concentrations. See, e.g., U.S. patent application Ser.
No. 08/337,915, filed Nov. 10, 1994 or International application
PCT/US95/14692, filed Nov. 10, 1995.
[0078] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques also well known in the art. Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1989) and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., most recent Supplement).
[0079] Nucleic acids used to transfect cells with sequences coding
for expression of the polypeptide of interest generally will be in
the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used herein, "operatively linked"
refers to a juxtaposition wherein the components so described are
in a relationship permitting them to function in their intended
manner. A control sequence operatively linked to a coding sequence
is ligated such that expression of the coding sequence is achieved
under conditions compatible with the control sequences. "Control
sequence" refers to polynucleotide sequences which are necessary to
effect the expression of coding and non-coding sequences to which
they are ligated. Control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence. The
term "control sequences" is intended to include, at a minimum,
components whose presence can influence expression, and can also
include additional components whose presence is advantageous, for
example, leader sequences and fusion partner sequences.
[0080] As used herein, the term "nucleotide sequence coding for
expression of" a polypeptide refers to a sequence that, upon
transcription and translation of mRNA, produces the polypeptide.
This can include sequences containing, e.g., introns. As used
herein, the term "expression control sequences" refers to nucleic
acid sequences that regulate the expression of a nucleic acid
sequence to which it is operatively linked. Expression control
sequences are operatively linked to a nucleic acid sequence when
the expression control sequences control and regulate the
transcription and, as appropriate, translation of the nucleic acid
sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons.
[0081] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the fluorescent
indicator coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. (See, for example,
the techniques described in Maniatis, et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).
Transformation of a host cell with recombinant DNA may be carried
out by conventional techniques as are well known to those skilled
in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0082] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransfected with DNA
sequences encoding the fusion polypeptide of the invention, and a
second foreign DNA molecule encoding a selectable phenotype, such
as the herpes simplex thymidine kinase gene. Another method is to
use a eukaryotic viral vector, such as simian virus 40 (SV40) or
bovine papilloma virus, to transiently infect or transform
eukaryotic cells and express the protein. (Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0083] Techniques for the isolation and purification of
polypeptides of the invention expressed in prokaryotes or
eukaryotes may be by any conventional means such as, for example,
preparative chromatographic separations and immunological
separations such as those involving the use of monoclonal or
polyclonal antibodies or antigen.
[0084] A variety of host-expression vector systems may be utilized
to express fluorescent indicator coding sequence. These include but
are not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing a fluorescent indicator coding sequence; yeast
transformed with recombinant yeast expression vectors containing
the fluorescent indicator coding sequence; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing a fluorescent indicator coding sequence; insect
cell systems infected with recombinant virus expression vectors
(e.g., baculovirus) containing a fluorescent indicator coding
sequence; or animal cell systems infected with recombinant virus
expression vectors (e.g., retroviruses, adenovirus, vaccinia virus)
containing a fluorescent indicator coding sequence, or transformed
animal cell systems engineered for stable expression.
[0085] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector (see, e.g., Bitter, et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
.lambda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
may be used. When cloning in mammalian cell systems, promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the retrovirus long
terminal repeat; the adenovirus late promoter; the vaccinia virus
7.5K promoter) may be used. Promoters produced by recombinant DNA
or synthetic techniques may also be used to provide for
transcription of the inserted fluorescent indicator coding
sequence.
[0086] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
fluorescent indicator expressed. For example, when large quantities
of the fluorescent indicator are to be produced, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Those which are
engineered to contain a cleavage site to aid in recovering
fluorescent indicator are preferred. In yeast, a number of vectors
containing constitutive or inducible promoters may be used. For a
review see, Current Protocols in Molecular Biology, Vol. 2, Ed.
Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience,
Ch. 13, 1988; Grant, et al., Expression and Secretion Vectors for
Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,
Acad. Press, N.Y., Vol. 153, pp.516-544, 1987; Glover, DNA Cloning,
Vol. II, IRL Pess, Wash., D.C., Ch. 3, 1986; and Bitter,
Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds.
Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684,
1987; and The Molecular Biology of the Yeast Saccharomyces, Eds.
Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982. A
constitutive yeast promoter such as ADH or LEU2 or an inducible
promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R.
Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M
Glover, IRL Press, Wash., D.C., 1986). Alternatively, vectors may
be used which promote integration of foreign DNA sequences into the
yeast chromosome.
[0087] In cases where plant expression vectors are used, the
expression of a fluorescent indicator coding sequence may be driven
by any of a number of promoters. For example, viral promoters such
as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al.,
Nature 310:511-514, 1984), or the coat protein promoter to TMV
(Takamatsu, et al., EMBO J. 6:307-311, 1987) may be used;
alternatively, plant promoters such as the small subunit of RUBISCO
(Coruzzi, et al., 1984, EMBO J. 3:1671-1680; Broglie, et al.,
Science 224:838-843, 1984); or heat shock promoters, e.g., soybean
hsp17.5-E or hsp17.3-B (Gurley, et al., Mol. Cell. Biol. 6:559-565,
1986) may be used. These constructs can be introduced into plant
cells using Ti plasmids, Ri plasmids, plant virus vectors, direct
DNA transformation, microinjection, electroporation, etc. For
reviews of such techniques see, for example, Weissbach &
Weissbach, Methods for Plant Molecular Biology, Academic Press, NY,
Section VIII, pp. 421-463, 1988; and Grierson & Corey, Plant
Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9, 1988.
[0088] An alternative expression system which could be used to
express fluorescent indicator is an insect system. In one such
system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The fluorescent indicator coding
sequence may be cloned into non-essential regions (for example, the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter). Successful
insertion of the fluorescent indicator coding sequence will result
in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed, see Smith, et al.,
J. Viol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051.
[0089] Eukaryotic systems, and preferably mammalian expression
systems, allow for proper post-translational modifications of
expressed mammalian proteins to occur. Eukaryotic cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, phosphorylation, and, advantageously
secretion of the gene product should be used as host cells for the
expression of fluorescent indicator. Such host cell lines may
include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,
Jurkat, HEK-293, and WI38. Primary cell lines, such as neonatal rat
myocytes, can also be used.
[0090] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the fluorescent indicator
coding sequence may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the fluorescent indicator
in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad.
Sci. USA, 81: 3655-3659, 1984). Alternatively, the vaccinia virus
7.5K promoter may-be used (e.g., see, Mackett, et al., Proc. Natl.
Acad. Sci. USA, 79: 7415-7419, 1982; Mackett, et al., J. Virol. 49:
857-864,-1984; Panicali, et al., Proc. Natl. Acad. Sci. USA 79:
4927-4931, 1982). Of particular interest are vectors based on
bovine papilloma virus which have the ability to replicate as
extrachromosomal elements (Sarver, et al., Mol. Cell. Biol. 1: 486,
1981). Shortly after entry of this DNA into mouse cells, the
plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration of
the plasmid into the host's chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such as
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of the fluorescent indicator gene in host cells (Cone
& Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353, 1984).
High level expression may also be achieved using inducible
promoters, including, but not limited to, the metallothionein IIA
promoter and heat shock promoters.
[0091] The recombinant nucleic acid can be incorporated into an
expression vector including expression control sequences
operatively linked to the recombinant nucleic acid. The expression
vector can be adapted for function in prokaryotes or eukaryotes by
inclusion of appropriate promoters, replication sequences, markers,
etc.
[0092] DNA sequences encoding the fluorescence indicator
polypeptide of the invention can be expressed in vitro or in vivo
by DNA transfer into a suitable recombinant host cell. As used
herein, "recombinant host cells" are cells in which a vector can be
propagated and its DNA expressed. The term also includes any
progeny of the subject host cell. It is understood that all progeny
may not be identical to the parental cell since there may be
mutations that occur during replication. However, such progeny are
included when the term "recombinant host cell" is used. Methods of
stable transfer, in other words when the foreign DNA is
continuously maintained in the host, are known in the art.
[0093] The expression vector can be transfected into a host cell
for expression of the recombinant nucleic acid. Recombinant host
cells can be selected for high levels of expression in order to
purify the fluorescent indicator fusion protein. E. coli is useful
for this purpose. Alternatively, the host cell can be a prokaryotic
or eukaryotic cell selected to study the activity of an enzyme
produced by the cell. In this case, the linker peptide is selected
to include an amino acid sequence recognized by the protease. The
cell can be, e.g., a cultured cell or a cell taken in vivo from a
transgenic animal.
[0094] Transgenic Animals
[0095] In another embodiment, the invention provides a transgenic
non-human animal that expresses a polynucleotide sequence which
encodes a fluorescent protein pH sensor.
[0096] The "non-human animals" of the invention comprise any
non-human animal having a polynucleotide sequence which encodes a
fluorescent indicator. Such non-human animals include vertebrates
such as rodents, non-human primates, sheep, dog, cow, pig,
amphibians, and reptiles. Preferred non-human animals are selected
from the rodent family including rat and mouse, most preferably
mouse. The "transgenic non-human animals" of the invention are
produced by introducing "transgenes" into the germline of the
non-human animal. Embryonal target cells at various developmental
stages can be used to introduce transgenes. Different methods are
used depending on the stage of development of the embryonal target
cell. The zygote is the best target for micro-injection. In the
mouse, the male pronucleus reaches the size of approximately 20
micrometers in diameter which allows reproducible injection of 1-2
pl of DNA solution. The use of zygotes as a target for gene
transfer has a major advantage in that in most cases the injected
DNA will be incorporated into the host gene before the first
cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442,
1985). As a consequence, all cells of the transgenic non-human
animal will carry the incorporated transgene. This will in general
also be reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene. Microinjection of zygotes is the preferred method
for incorporating transgenes in practicing the invention.
[0097] The term "transgenic" is used to describe an animal which
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by cross-breeding two chimeric
animals which include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic, i.e., animals which include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0098] Retroviral infection can also be used to introduce transgene
into a non-human animal. The developing non-human embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retro viral infection (Jaenisch, R.,
Proc. Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection
of the blastomeres is obtained by enzymatic treatment to remove the
zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.). The viral vector system used to introduce the transgene is
typically a replication-defective retrovirus carrying the transgene
(Jahner, et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van
der Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Van der
Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele (D.
Jahner et al., Nature 298:623-628, 1982). Most of the founders will
be mosaic for the transgene since incorporation occurs only in a
subset of the cells which formed the transgenic nonhuman animal.
Further, the founder may contain various retro viral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retro viral infection of the
midgestation embryo (D. Jahner et al., supra). A third type of
target cell for transgene introduction is the embryonal stem cell
(ES). ES cells are obtained from pre-implantation embryos cultured
in vitro and fused with embryos (M. J. Evans et al. Nature
292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258,1984;
Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; and
Robertson et al., Nature 322:445-448, 1986). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a nonhuman animal. The
ES cells thereafter colonize the embryo and contribute to the germ
line of the resulting chimeric animal. (For review see Jaenisch,
R., Science 240: 1468-1474, 1988).
[0099] "Transformed" means a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous polynucleotide. "Heterologous" refers to
a polynucleotide sequence that either originates from another
species or is modified from either its original form or the form
primarily expressed in the cell.
[0100] "Transgene" means any piece of DNA which is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) which develops from that-cell. Such a
transgene may include a gene which is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence which is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode the fluorescent indicator which
may be expressed in a transgenic non-human animal. The term
"transgenic" as used herein additionally includes any organism
whose genome has been altered by in vitro manipulation of the early
embryo or fertilized egg or by any transgenic technology to induce
a specific gene knockout. The term "gene knockout" as used herein,
refers to the targeted disruption of a gene in vivo with complete
loss of function that has been achieved by any transgenic
technology familiar to those in the art. In one embodiment,
transgenic animals having gene knockouts are those in which the
target gene has been rendered nonfunctional by an insertion
targeted to the gene to be rendered non-functional by homologous
recombination. As used herein, the term "transgenic" includes any
transgenic technology familiar to those in the art which can
produce an organism carrying an introduced transgene or one in
which an endogenous gene has been rendered non-functional or
"knocked out."
[0101] Detection of pH Using Fluorescent Indicator Proteins
[0102] In another embodiment, the invention provides a method for
determining the pH of a sample by contacting the sample with an
indicator including a first fluorescent protein moiety whose
emission intensity changes as pH varies between pH 5 and 10,
exciting the indicator, and then determining the intensity of light
emitted by the first fluorescent protein moiety at a first
wavelength. The emission intensity of the first fluorescent protein
moiety indicates the pH of the sample.
[0103] The fluorescent protein moiety can be a functional
engineered protein substantially identical to the amino acid
sequence of Aequora green fluorescence protein (SEQ ID NO: ______).
Preferred green fluorescence proteins include those having a
functional engineered fluorescent protein that includes one of the
following sets of substitutions in the amino acid sequence of the
Aequora green fluorescent protein (SEQ ID NO:______):
S65G/S72A/T203Y/H231L, S65G/V68L/Q69K/S72A/T203Y, or
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H- /H231L. Other
preferred green fluorescence proteins include those having a
functional engineered fluorescent protein that includes H148G or
H148Q substitutions in the Aequora green fluorescent protein. These
proteins include the YFP H148G (SEQ ID NO:______) and YFP H148Q
(SEQ ID NO: ______) proteins described above.
[0104] The sample in which pH is to be measured can be a biological
sample, e.g., a biological tissue such as an extracellular matrix,
blood or lymphatic tissue, or a cell. The method is particularly
suitable for measuring pH in a specific region of the cell, e.g.,
the cytosol, or an organellar space such as the inner mitochondrial
matrix, the lumen of the Golgi, cytosol, the endoplasmic reticulum,
the chloroplast lumen, the lumen of lysosome, or the lumen of an
endosome.
[0105] In some embodiments, the first fluorescent protein moiety is
linked to a targeting sequence that directs the fluorescent protein
to a desired cellular compartment. Examples of targeting sequences
include the amino terminal 81 amino acids of human type II
membrane-anchored protein galactosyltransferase (SEQ ID NO:______)
for directing the fluorescent indicator protein to the Golgi and
the amino terminal 12 amino acids of the presequence of subunit IV
of cytochrome c oxidase (SEQ ID NO:______) for directing a
fluorescent pH indicator protein to the mitochondrial matrix. The
12 amino acids of the presequence of subunit IV of cytochrome c
oxidase (SEQ ID NO:______) may be linked to the pH fluorescent
indicator protein through a linker sequence, e.g., Arg-Ser-Gly-Ile
(SEQ ID NO:______).
[0106] In another embodiment, the invention provides a method of
determining the pH of a region of a cell by introducing into the
cell a polynucleotide encoding a polypeptide including an indicator
having a first fluorescent protein moiety whose emission intensity
changes as pH varies between 5 and 10, culturing the cell under
conditions that permit expression of the polynucleotide; exciting
the indicator; and determining the intensity of the light emitted
by the first protein moiety at a first wavelength. The emission
intensity of the first fluorescent protein moiety indicates the pH
of the region of the cell in which the indicator is present.
[0107] The polynucleotide can be introduced using methods described
above. Thus, the method can be used to measure intracellular pH in
cells cultured in vitro, e.g., HeLa cells, or alternatively in
vivo, e.g., in cells of an animal carrying a transgene encoding a
pH-dependent fluorescent indicator protein.
[0108] Fluorescence in the sample can be measured using a
fluorometer. In general, excitation radiation, from an excitation
source having a first wavelength, passes through excitation optics.
The excitation optics cause the excitation radiation to excite the
sample. In response, fluorescent proteins in the sample emit
radiation which has a wavelength that is different from the
excitation wavelength. Collection optics then collect the emission
from the sample. The device can include a temperature controller to
maintain the sample at a specific temperature while it is being
scanned. If desired, a multi-axis translation stage can be used to
move a microtiter plate holding a plurality of samples in order to
position different wells to be exposed. The multi-axis translation
stage, temperature controller, auto-focusing feature, and
electronics associated with imaging and data collection can be
managed by an appropriately programmed digital computer. The
computer also can transform the data collected during the assay
into another format for presentation.
[0109] Methods of performing assays on fluorescent materials are
well known in the art and are described in, e.g., Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York:Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern
Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing
Col, Inc. (1978), pp. 296-361.
[0110] The pH can be analyzed on cells in vivo, or from samples
derived from cells transfected with polynucleotides or proteins
expressing the pH indicator proteins. Because fluorescent pH
indicator proteins can be expressed recombinantly inside a cell,
the pH in an intracellular region, e.g., an organelle, or an
extracellular region of an organism can be determined simply by
determining changes in fluorescence.
[0111] Fluorescent protein pH sensors may vary in their respective
pK.sub.a, and the differences in pK.sub.a can be used to select the
most suitable fluorescent protein sensor most suitable for a
particular application. In general, a sensor protein should be used
whose pK.sub.a is close to the pH of the sample to be measured.
Preferably the pK.sub.a is within 1.5 pH unit of the sample. More
preferably the pK.sub.a is within 1 pH unit, and still more
preferably the pK.sub.a is within 0.5 pH unit of the sample.
[0112] Thus, a fluorescent protein pH sensor having a pK.sub.a of
about 7.1, e.g., the EYFP mutant described below, is preferred for
determining the pH of cytosolic, Golgi, and mitochondrial matrix pH
areas of a cell. The YFP-H148G, YFP-H148Q, EYFP-H148G and
EYFP-H148Q mutants are well-suited for measuring the pH of alkaline
environments, e.g., mitochondrial matrix, as they have a pKa of 7.5
and 8.0, respectively.
[0113] For more acidic organelles, a fluorescence sensor protein
having a lower pK.sub.a, e.g., a pK.sub.a of about 6.1, is
preferred.
[0114] To minimize artefactually low fluorescence measurements that
occur due to cell movement or focusing, the fluorescence of a
fluorescent protein pH sensor can be compared to the fluorescence
of a second sensor, e.g., a second fluorescent protein pH sensor,
that is also present in the measured sample. The second fluorescent
protein pH sensor should have an emission spectra distinct from the
first fluorescent protein pH sensor so that the emission spectra of
the two sensors can be distinguished. Because experimental
conditions such as focusing and cell movement will affect
fluorescence of the second sensor as well as the first sensor,
comparing the relative fluorescence of, the two sensors allows for
the normalization of fluorescence.
[0115] A convenient method of comparing the samples is to compute
the ratio of the fluorescence of the first fluorescent protein pH
sensor to that of the second fluorescent protein pH sensor.
[0116] Kits
[0117] The materials and components described for use in the
methods of the invention are ideally suited for the preparation of
a kit. Such a kit may comprise a carrier means being
compartmentalized to receive one or more container means such as
vials, tubes, and the like, each of the container means comprising
one of the separate elements to be used in the method. For example,
one of the container means may comprise a polynucleotide encoding a
fluorescent protein pH sensor. A second container may further
comprise fluorescent protein pH sensor. The constituents may be
present in liquid or lyophilized form, as desired.
[0118] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to limit the scope
of the invention described in the claims.
EXAMPLES
Example 1
Construction of Fluorescent Protein pH Sensors
[0119] Fluorescent protein pH sensors were constructed by
engineering site-specific mutations in polynucleotides encoding
forms of the Aequora victoria green fluorescent protein (GFP). The
starting GFP variant was the polynucleotide encoding the GFP
variant EGFP (for enhanced green fluorescent protein). The EGFP
variant had the amino acid substitutions F64L/S65T/H231L relative
to the wild-type Aequora victoria GFP sequence.
[0120] The ECFP (enhanced cyan fluorescent protein) mutant was
constructed by altering the EGFP polynucleotide sequence so that it
encoded a protein having the amino acid substitutions
K26R/F64L/S65T/Y66W/N146I/M153T/V163A- /N164H/H231L relative to the
wild-type GFP amino acid sequence. A second variant, named EYFP
(enhanced yellow fluorescent protein) was constructed by altering
the EGFP polynucleotide to encode a protein having the amino acid
substitutions S65G/S72A/T203Y/H231L relative to the amino acid
sequence of GFP. A third variant, named EYFP-V68L-Q69K, was
constructed by altering the EGFP polynucleotide to encode a protein
having the amino acid substitutions S65G/V68L/Q69K/S72A/T203Y
relative to the amino acid sequence of GFP.
[0121] A HindIII site and Kozak consensus sequence (GCCACCATG) was
introduced at the 5' end of the polynucleotide encoding the GFP
variants, and an EcoR1 site was added at the 3' end of the gene of
each indicator, and the fragments were ultimately ligated into the
HindIII/EcoR1 sites of the mammalian expression vector pcDNA3
(Invitrogen). EGFP and EYFP mutant proteins with no targeting
signals were used as indicators of pH in the cytosol or
nucleus.
[0122] To construct fluorescent protein pH sensors to use as pH
indicators in the Golgi, polynucleotides encoding the 81 N-terminal
amino acids of the type II membrane-anchored protein
galactosyltransferase
(GT:UDP-galactose-.beta.,1,4-galactosyltransferase. EC 2.4.1.22)
ligated to polynucleotides encoding EGFP, ECFP, or EYFP. The
polynucleotides encoding the resulting proteins were named GT-EGFP,
GT-ECFP, and GT-EYFP, respectively.
[0123] Mitochondrial matrix fluorescent protein pH sensors were
constructed by attaching polynucleotides encoding 12 amino acids at
the amino terminus of the presequence of subunit IV of cytochrome c
oxidase (Hurt et al, EMBO J. 4:2061-68 (1985) to a polynucleotide
encoding the amino acid sequence Arg-Sea-Gly-Ile, which in turn was
ligated to polynucleotides encoding ECFP or EYFP. These constructs
were labeled ECFP-mito or EYFP-mito.
[0124] The constructs used to examine intracellular pH are
summarized in FIG. 1.
Example 2
pH Titration of Fluorescent Sensor Proteins in Vitro
[0125] The pH sensitivity of the fluorescence of the proteins ECFP,
EGFP, EYFP, GT-EGFP, and GT-EYFP was first examined.
[0126] Absorbance spectra were obtained in a Cary 3E
spectrophotometer (Varian). For pH titration, a
monochromator-equipped fluorometer (Spex Industries, NJ) and a
96-well microplate fluorometer (Cambridge Technology) were used. In
the latter case the filters used for excitation were 482.+-.10
(460.+-.18 for ECFP) and for emission were 532.+-.14. Filters were
named as the center wavelength.+-.the half-bandwidth, both in nm.
The solutions for cuvette titration contained 125 mM KCl, 20 mM
NaCl, 0.5 mM CaCl.sub.2, 0.5 mM MgCl.sub.2, and 25 mM of one of the
following buffers--acetate, Mes, Mops, Hepes, bicine, and Tris.
[0127] EYFP showed an acidification-dependent decrease in the
absorbance peak at 514 nm and a concomitant increase in absorbance
at 390 nm (FIG. 2A). The fluorescence emission (527-nm peak) and
excitation spectra decreased with decreasing pH, but the
fluorescence excitation spectrum showed no compensating increase at
390 nm. Therefore, the species absorbing at 390 nm was
nonfluorescent. The apparent pKa (pK'a) of EYFP was 7.1 with a Hill
coefficient (n) of 1.1 (FIG. 2B)
[0128] EGFP fluorescence also was quenched with decreasing pH. The
pK'a of EGFP was 6.15, and n was 0.7.
[0129] The change in fluorescence of ECFP (Tyr66.fwdarw.Trp in the
chromophore) with pH was smaller than that of EGFP or EYFP (pK'a
6.4, n, 0.6) (FIG. 2B) The fluorescence change was reversible in
the pH range 5-8.5 for all three proteins, which covers the pH
range of most subcellular compartments. These results demonstrate
that the GFP variants EGFP, EYFP, and ECFP can be used as
fluorescent protein pH sensors.
Example 3
Measurements of pH in the Cytosol and Nucleus Using Fluorescent
Protein pH Sensors
[0130] HeLa cells and AT-20 cells grown on glass coverslips were
transiently lipo-transfected (Lipofectin.TM., GIBCO) with
polynucleotide constructs encoding EYFP.
[0131] Cells were imaged between 2 and 4 days after transfection at
22.degree. C. with a cooled charge-coupled device camera
(Photometrices, Tucson, Ariz.) as described in Miyawaki et al.,
Nature 388:882, (1997). The interference filters (Omega Optical and
Chroma Technology, Brattleboro, Vt.) used for excitation and
emission were 440.+-.10 and 480.+-.15 for ECFP; 480.+-.15 and
535.+-.22.5 for EGFP or EYFP. The dichroic mirrors were 455 DCLP
for ECFP and 505 DCLP for EGFP or EYFP. Regions of interest were
selected manually, and pixel intensities were spatially averaged
after background subtraction. A binning of 2 was used to improve
signal/noise and minimize photodamage and photoisomerization of
EYFP. High KCl buffer plus 5 .mu.M each of the ionophores nigericin
(Fluka) and monensin (Calbiochem) was used for in situ titrations
in living cells. Cells were loaded with cytosolic pH indicators by
incubation with 3 .mu.M carboxy-SNARF/AM or BCECF/AM (Molecular
Probes) for 45 minutes, then washed for 30 minutes, all at
22.degree. C.
[0132] Fluorescence of HeLa cells transfected with the gene
encoding EYFP was diffusely distributed in the cytosol and nucleus.
This was expected for a protein of the size of GFP (27 kDa), which
is small enough to pass through nuclear pores.
[0133] The fluorescence observed with EYFP was reversible.
Perfusion with NH.sub.4Cl caused an increase in fluorescence (rise
in pH), which reversed upon washing out the NH.sub.4Cl. Conversely,
perfusion of lactate, which lowers pH, induced a decrease in
fluorescence. The decrease in fluorescence was also reversible on
wash-out.
[0134] Calibration of fluorescence intensity with pH in situ was
accomplished with a mix of the alkali cation/H+ ionophores
nigericin and monensin in bath solutions of defined pH and high K+.
Fluorescence equilibrated within 1-4 minutes after each exchange of
solution. These results demonstrate that EYFP, when present
intracellularly, can report pH in the physiological range.
Example 4
Measurement of pH in the Mitochondrial Matrix Using Fluorescent
Protein pH Sensors
[0135] To measure pH in the mitochondrial matrix using mutant GFP
sensor proteins, HeLa cells and neonatal rat cardiomyocytes were
transfected with the fluorescent protein pH sensor EYFP-mito. A
Bio-Rad MRC-1000 confocal microscope was used for analysis of the
targeted protein. Microscopy analysis revealed that the transfected
cells showed a fluorescence pattern indistinguishable from that of
the conventional mitochondrial dye rhodamine 123.
[0136] In situ pH titration was performed with nigericin/monensin
as described in Example 3. Subsequent addition of the protonophore
carbonylcyanide m-chlorophenylhydrazone (CCCP) did not change the
fluorescence intensity of the cells. This demonstrates that the
nigericin/monensin treatment effectively collapsed the pH gradient
(.DELTA.pH) in the mitochondria.
[0137] The estimated pHm was 7.98.+-.0.07 in HeLa cells (n=17
cells, from six experiments). Similar pH values were obtained in a
HeLa cell line stably expressing EYFP-mito. Resting pH did not
change by superfusion of cells with medium 10 mM glucose, which
would provide cells with an oxidizable substrate, but 10 mM lactate
plus 1 mM pyruvate caused an acidification, which reversed on
washout. This can be accounted for by diffusion of protonated acid
or by cotransport of pyruvate.sup.-/H.sup.+ through the inner
mitochondrial membrane. The protonophore CCCP rapidly induced an
acidification of mitochondria to about pH 7.
Example 5
Measurement of pH in the Golgi Lumen Using Fluorescent Protein pH
Sensors
[0138] The type II membrane-anchored protein galactosyltransferase
(GT:UDP-galactose-.beta.,1,4-galactosyltransferase. EC 2.4.1.22)
has been used as a marker of the trans cisternae of the Golgi
apparatus (Roth et al., J. Cell Biol. 93:223-29, (1982)).
Accordingly, polynucleotide constructs encoding portions of the GT
protein fused to the mutant GFP proteins were constructed as
described in Example 1 in order to use the GT sequence to target
the fluorescent protein pH sensor to the endoplasmic reticulum.
[0139] The pH of the Golgi lumen was measured by transfecting HeLa
or AT-20 cells with the constructs GT-ECFP, GT-EGFP, or GT-EYFP.
Bright juxtanuclear fluorescence was observed, with little increase
in diffuse staining above autofluorescnce in most cells.
[0140] The fluorescence pattern was examined further in
double-labeling experiments using rabbit polyclonal
.alpha.-mannosidase II (.alpha.-manII) antibody. Double labeling
fluorescence was performed as described by McCaffery et al.,
Methods Enzymol. 257:259-279 (1995). The .alpha.-manII antibody was
prepared as described in Velasco et al., J. Cell Biol. 122:39-51
(1993). In the double-staining experiments, it was observed that
labeling of the medial trans-Golgi marker .alpha.-manII overlapped
with GT-EYFP fluorescence.
[0141] .alpha.-manII was also fused with ECFP, and the pattern of
fluorescence obtained upon transfection of the gene was
indistinguishable from that of GT-EYFP by light microcopy.
[0142] To identify the subcellular localization of GT-EYFP at
higher resolution, immunogold electron microscopy was performed on
ultra-thin cryosections by using antibodies against GFP. Immunogold
labeling of ultra-thin sections was performed as described by
McCaffery et al., supra, using rabbit polyclonal anti-GFP antibody
or a monoclonal anti-TGN38 antibody.
[0143] In double-labeling experiments, GT-EYFP was found in the
medial and trans Golgi, although endogenous GT is present in trans
Golgi membranes. The difference in localization may occur as a
result of overexpression of the GT-EYFP protein.
[0144] When protein TGN38 was used as a trans-Golgi network (TGN)
marker, its immunogold localization pattern was found to overlap
with that of GT-EYFP in the medial/trans-Golgi membranes. The
localization data demonstrate that GT-EYFP labels the medial/trans
Golgi. Thus, GT-EYFP can be used to identify the pH of this
organelle.
[0145] The pH titration of GT-EYFP fluorescence in the Golgi region
of the cells after treatment with nigericin/monensin was in good
agreement with that of EYFP in vitro (see Example 2). Resting pH in
HeLa cells was on average 6.58 (range 6.4-6.81, n=30 cells, 9
experiments). These results also demonstrate that-neither fusion
with GT nor the composition of the Golgi lumen affects the pH
sensitivity of EYFP. Thus, Golgi-targeted EYFP can be used as a
local pH indicator.
[0146] The effect of various treatments on the pH of the Golgi was
next examined using Golgi-targeted EYFP.
[0147] The pH gradient across the Golgi membrane is maintained by
the electrogenic ATP-dependent H.sup.+ pump (V-ATPase). The
V-ATPase generates a .DELTA.pH (acidic inside) and .DELTA..psi.
(positive inside), which opposes further H.sup.+ transport. The
movement of counter-ions, Cl.sup.- in (or K.sup.+ out), with
H.sup.+ uptake would shunt the .DELTA..psi., allowing a larger
.DELTA.pH to be generated. These mechanisms were investigated in
intact single HeLa cells transfected with GT-EYFP.
[0148] The macrolide antibiotic bafilomycin A1 has been shown to be
a potent inhibitor of vacuolar type H.sup.+ ATPases (V type). In
Hela cells expressing GT-EYFP, bafilomycin A1 (0.2 .mu.M) increased
pH.sub.G by about 0.6 units, to pH 7.16 (range 7.02-7.37, n=12
cells. This suggests that the H.sup.+ pump compensates for a
positive H+ efflux or leak. The initial rate of Golgi
alkalinization by bafilomycin A1 was 0.52 pH units per minute
(range 0.3-0.77, n=12 cells), faster than that reported for other
acidic compartments such as macrophage phagosomes (0.09 pH/min).
Similar results regarding resting pHG and alkalinization by
bafilomycin Al were obtained when HeLa cells were transfected with
GT-EGFP. Calibration of GT-EGFP in situ also mirrored its in vitro
titration (FIG. 1B). Thus, both EGFP and EYFP are suitable Golgi pH
indicators.
Example 6
Measuring Intracellular pH with Two Fluorescent Protein Sensors
[0149] Quantitative measurements of fluorescence with
nonratiometric indicators can suffer from artifacts as a result of
cell movement or focusing. To correct for these effects, the
cyan-emitting mutant GT-ECFP was co-transfected into cells along
with GT-EYFP. ECFP has excitation and emission peaks that can be
separated from those of EYFP by appropriate filters. In addition,
ECFP is less pH-sensitive than EYFP (see FIG. 2B).
[0150] FIG. 3A demonstrates that the fluorescence of ECFP changed
less than that of EYFP during the course of the experiment.
Although the ratio of EYFP to ECFP emission varied between cells,
probably reflecting a different concentration of GT-EYFP and
GT-ECFP in the Golgi lumen, it changed with pH as expected (FIG.
3B). Bafilomcin A1 raised the GT-EYFP/GT-ECFP emission ratio, i.e,
it raised pH.sub.G.
Example 7
Construction of YFP THR H148G and YFP H1480 Mutants
[0151] The YFP H148G mutant was prepared using as a template a
nucleic acid encoding the YFP mutation 10c, which includes the
mutations S65G/V68L/S72A/Q80R/T203Y and is described in Ormo et
al., Science 273:1392-95 (1997). The YFP H148G mutant was
constructed using the PCR-based QUIKCHANGE.TM. Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, Calif.) following the
manufacturer's instructions. The YFP H148Q mutation was similarly
constructed from a nucleic acid encoding the 10C mutation.
[0152] The pKa of the YFP H148G mutant was found to be 8.0, while
the YFP H148Q mutant was found to have a pKa of 7.5.
Example 8
Expression of Mito-YFP H148G in the Mitochondrial Matrix at a pH
Range of 7.0 to 8.4
[0153] The high pKa of the mutant YFP H148G allows it the to be
used for the precise measurement of mitochondrial matrix pH both in
cells at rest and in cells subject to manipulations that decrease
mitochondrial pH.
[0154] This was demonstrated directly by transfecting a nucleic
acid encoding mito-YFP H148G into HeLa cells using the procedures
described in Example 4. YFP H148G expression was monitored by
observing fluorescence over time. Mitochondrial pH was also
monitored by pH titration as described in Example 3 using nigericin
and monensin.
[0155] FIG. 4 shows that HeLa cells transfected with YFP H148G in
the mitochondrial matrix were fluorescent at an initial pH of 8.0
to 8.1 (where measurements began at t.apprxeq.0 seconds). 5 .mu.M
CCCp was added at about t.apprxeq.300 seconds. Although addition of
5 .mu.m CCCP rapidly lowered the pH to 7.0, fluorescence of
mito-YFP H148G was still detectable. Then a calibration was
performed by perfusing the cells with extracellular medium of ph 7,
7.5, 8, and 8.35 containing the ionophores nigericin plus monensin
to equilibrate mitochondrial pH and extracellular pH. Fluorescence
in mitochondria increased stepwise with each change of
extracellular pH.
[0156] Fluorescence was also examined, and pH measured, in primary
cultures of chick skeletal myotubes transfected with the mito-YFP
H148G mutant. FIG. 5 demonstrates that fluorescence was detectable
in the mitochondrial matrix of chicken skeletal myotubes, which had
a pH of 8.0-8.1 (t.apprxeq.0). Fluorescence was still detectable
following addition of 25 .mu.M forskolin, which did not affect the
pH, and after addition of 2 .mu.M CCCP at t.apprxeq.750 seconds,
although CCCP caused the pH to rapidly drop to 6.9 at
t.apprxeq.1400 seconds. Thereafter fluorescence continued to be
observed during calibration at ph 6.9, 7.6 and 8.0.
[0157] These results demonstrate mito-YFP H148G fluorescence is
detectable in the mitochondrial matrix over the pH range of 7.0 to
8.4 in both established cell lines (HeLa cells) and primary
cultures (chick skeletal myotubes).
Example 9
Expression of Mito-YFP H148Q in Response to PH Changes
[0158] The YFP H148Q mutant has a pKa of about 7.4, which is
intermediate between the pKa of EYFP and YFP mutant H148G. To
demonstrate that YFP H148Q can also be used to measure
mitochondrial matrix pH, a nucleic acid encoding mito-YFP-H148Q was
transfected into HeLa cells. Fluorescence was measured over time
(beginning at t.apprxeq.0), including following the addition of 10
.mu.M nigericin in high KCL titration buffer at t.apprxeq.500
seconds.
[0159] FIG. 6 reveals the effect of changing mitochondrial pH to
6.9, 7.5, 8, and 8.4 with the ionophore nigericin on fluorescence
intensity. Fluorescence decreased to about 175 units at t=1000
seconds by addition of nigericin, which lowered the pH to about
6.9. Fluorescence then returned stepwise to 400 units with each
change of the extracellular medium. These results demonstrate that
the fluorescence of the mito-YFP H148Q mutant can be used to
measure the pH of the inner mitochondrial matrix.
Example 10
Measuring Intracellular pH by Coexpression of YFP H148G and a
Second PH-Insensitive Sensor
[0160] As is discussed above in Example 6, for quantitative
measurements it is desirable to compute the fluorescence of the
sensor used to measure pH with the fluorescence of a second sensor
molecule whose fluorescence does not change over the pH range being
tested. A ratiometric measurement is useful to correct for movement
or focusing artifacts that may occur during live cell imaging
experiments.
[0161] To identify a GFP sensor protein suitable for use as a
reference protein for measuring mitochondrial matrix pH, the GFP
mutant T203I was expressed in the mitochondria of HeLa cells. The
GFP T203I mutant can be excited with light of 400 nm, which does
not appreciably excite the pH sensitive YFP mutants.
[0162] Fluorescence of HeLa cells transfected with the GFP T2031
mutant was monitored for about 400 seconds using an excitation
ratio of 480 nm/400 nm. 10 .mu.M CCCP was then added to the cells,
and fluorescence was monitored for an additional 250 seconds.
Addition of CCCP did not affect fluorescence. In control
experiments, it was observed that addition of CCCP corresponded to
a drop in pH of about 1 unit. Thus, the GFP T203I mutant is
suitable for use as a reference, pH-insensitive mutant.
[0163] HeLa cells were then transfected with the GFP T203I mutant
and YFP H148G. FIG. 7 shows the change of mitochondrial pH with
oligomycin and the uncoupler CCCP as the ratio of YFP H148G
emission and GFP T203I emission, with excitation of 490 and 400 nm,
respectively.
Example 11
Structural Characterization of YFP T203Y/S65G/V68L/S72A/H148G
[0164] The green fluorescent protein (GFP) from the jellyfish
Aequorea victoria has been used extensively in molecular biology as
a fluorescent label. The structures of WT GFP (Yang et al., Nature
Biotech. 14:1246-51, 1996; Brejc et al., Proc. Natl. Acad. Sci.
USA. 94:2306-11, 1997) and the variant S65T were determined in 1996
(Ormo et al., Science 273:1392-95, 1996).
[0165] A large number of mutants have been identified that exhibit
broadly varying absorption and emission maxima (Heim et al. above;
Heim et al., Curr. Biol. 6: 178-82, 1996). The yellow fluorescent
protein (YFP) mutant is of particular interest since its spectrum
is shifted enough to render it readily distinguishable from the
spectrum of Cyan Fluorescent Protein (CFP) for FRET measurements
(Tsien, Ann. Rev. Biochem. 67:509, 1998; Miawaki et al., Nature
388:882-87, 1997). WT GFP exhibits two absorption maxima, where the
major band absorbs at 398 nm and the minor band at 475 nm (Morise
et al., Biochemistry 13:2656-62, 1974). Excitation of either of
these bands leads to emission of green light with a maximum between
504 and 508 nm (FIG. 8). Before a structure was available, GFP
variants with altered spectral characteristics were identified by
random mutagenesis. Some of these mutants, such as Y66H and Y66W
(Tsien, Ann. Rev. Biochem. 67:509, 1998, Heim et al., Proc. Natl.
Acad. Sci. (USA) 91:12501-04) result in blue-shifted absorbance and
emission maxima. Others focus on changes in the immediate
environment of the chromophore .pi. system, such as S65T (Heim et
al., Nature 373: 663-64, 1995) and T203I (Heim et al., Proc. Natl.
Acad. Sci. (USA) 91:12501-04, 1994). At physiological pH, S65T
exhibits only one major absorption band at 489 nm, red-shifted by
14 nm from WT GFP, and is almost six times brighter (Heim et al.,
Nature 373: 663-64, 1995). Yet, the emission spectrum is shifted by
only 3 nm to 511 nm, and so cannot easily be distinguished from the
wild-type emission. Random mutagenesis techniques produced only one
further red-shifted variant, S65T/M153A/K238E, which increases the
excitation and emission wavelengths of S65T by 15 and 3 nm
respectively (Heim et al., Curr. Biol. 6: 178-82, 1996). Here is
described crystal structures of the first set of GFP variants
rationally designed based on the x-ray structure of GFP S65T (Ormo
et al., Science 273:1392-95, 1996). These variants, termed YFPs
(Yellow Fluorescent Proteins), exhibit the longest wavelength
emissions of all GFPs generated by mutagenesis (FIG. 8). The YFPs
fluoresce around 528 nm, red-shifted by 16 nm as compared to S65T
and are easily distinguishable from S65T on a fluorescence
microscope.
[0166] The specific YFP investigated is the quadruple-mutant
T203Y/S65G/V68L/S72A, where the substitution T203Y was introduced
based on the structural considerations detailed below and is
believed responsible for the red-shift. The other three mutations
have been shown to improve its brightness in live cells (Cormack et
al., Gene 173:33, 1996). The T203Y mutation would have been
difficult to identify by random mutagenesis since this amino acid
substitution requires three substitutions at the nucleotide level.
Since Thr203 is positioned close to the chromophore, it was
postulated that its replacement with a tyrosine would result in
.pi.-stacking interactions between the chromophore and the highly
polarizable phenol (Ormo et al., Science 273:1392-95, 1996),
leading to red-shifted spectral properties. The structure of S65T
suggested that an aromatic amino acid introduced in position 203
would extend into the water-filled cavity adjacent to the
chromophore (Ormo et al., Science 273:1392-95, 1996). Replacement
of Thr203 with any of the aromatic amino acids His, Trp,Tyr, or Phe
was found to lead to the desired spectral shifts (Ormo et al.,
Science 273:1392-95, 1996, Dickson et al., Nature 388:355-58,
1997). The most dramatic red-shift was observed for the T203Y
substitution, therefore this variant has been termed YFP.
[0167] In order to determine the role of His148 in modulating the
pKa of the chromophore or its spectral properties, an additional
mutation, H148G, was introduced into the YFP background. The x-ray
structures of YFP and YFP H148G were analyzed in order to better
correlate structural changes with spectral properties. GFP variants
were prepared as described in Example 6, above. This template
incorporates the mutations T203Y/S65G/V68L/S72A, as well as the
ubiquitous Q80R substitution that was accidentally introduced into
the gfp cDNA early on (Ormo et al., Science 273:1392-95, 1996;
Chalfie et al., Science 263:802-05, 1994). All GFP variants were
expressed and purified as described (Ormo et al., Science
273:1392-95, 1996).
[0168] Structural Determination of YFP H148G
[0169] YFP H148G was concentrated to 12 mg/ml in 20 mM HEPES pH
7.9. Rod-shaped crystals with approximate dimensions of
1.8.times.0.08.times.0.04 mm were grown in hanging drops containing
2 .mu.l protein and 2 .mu.l mother liquor at 4.degree. C. within
four days. The mother liquor contained 16% PEG 4000, 50 mM sodium
acetate pH 4.6, and 50 mM ammonium acetate. X-ray diffraction data
were collected from a single crystal at room temperature using a
Xuong-Hamlin area detector (Hamlin, Methods. Enzymol. 114:416-52,
1985). Data were collected in space group P2.sub.12.sub.12.sub.1,
to 99% completeness at 2.6 .ANG. resolution, and reduced using the
supplied software (Howard et al., Methods Enzymol. 114:452-71,
1985). Unit cell parameters were a=52.0, b=62.7, and c=69.9. The
GFP S65T coordinate file (Ormo et al., Science 273:1392-95, 1996)
which-served as a model for phasing was edited to reflect the
mutations, with the introduced residues Tyr203 and Leu68 initially
modeled as alanines to prevent model bias. A model for the anionic
chromophore was obtained by semi-empirical molecular orbital
calculations using AM1 in the program SPARTAN version 4.1
(Wavefunction Inc., Irvine, Calif.). The minimized structure, which
was planar, compared very favorably with a related small molecule
crystallographic structure (Tinant et al., Cryst. Struct. Comm.
9:671-74, 1980), and also with the model used during refinement of
GFP S65T, where a simpler modeling program had been employed (Ormo
et al., Science 273:1392-95, 1996).
[0170] Using the program TNT (Tronrud et al., Acta Crystallogr.
Sect. A 43:489, 1987), rigid body refinement was carried out to
position the isomorphous model in the unit cell of YFP H148G.
Initial positional refinement was carried out using the data to 4.0
.ANG., then to 3.5, 3.0, and finally to 2.6 .ANG.. Electron density
maps (2Fo-Fc and Fo-Fc) were inspected using O (Tronrud et el.,
above), and solvent molecules were added if consistent with Fo-Fc
features, and only when in proximity of hydrogen bond partners.
B-factors were refined using a strong correlation between
neighboring atoms due to the relatively low resolution. Since no
B-factor library is available for the chromophore itself, the
B-factors of all chromophore atoms were set to the values obtained
in the 1.9 .ANG. structure of GFP S65T (Ormo et al., Science
273:1392-95, 1996), and then refined as a group, with identical
shifts for the grouped atoms.
[0171] Structure Determination of YFP
[0172] YFP was concentrated to 10 mg/ml in 50 mM HEPES pH 7.5.
After 2 weeks crystals grew to a size of 0.03.times.0.12.times.0.8
mm at 15.degree. C. in hanging drops containing 5=.vertline.l
protein and 5=.vertline.l well solution, which contained 2.2 M
sodium/potassium phosphate pH 6.9. These crystals belong to space
group P2.sub.12.sub.12 and have the unit cell dimensions a=77.1,
b=117.4, w and c=62.7. X-ray diffraction data were collected on two
isomorphic crystals at room temperature using an Raxis-IV imaging
plate mounted on a Rigaku RUH3 rotating anode generator equipped
with mirrors. The data were processed with Denzo and scaled using
ScalePack (Otwinowski et al., Methods Enzymol. 276:307-26, 1997).
The YFP structure was solved by molecular replacement using the
program AMoRe (Navaza, Acta Crystallogr. A50:157-63, 1994), with
the 1.9 .ANG. GFP S65T coordinate file as the search model (Ormo et
al., Science 273:1392-95, 1996). Two solutions were identified,
consistent with two molecules per asymmetric unit.
[0173] For refinement, the 2.6 .ANG. structure of YFP H148G was
chosen as the initial model, which was edited to reflect the
mutations present in YFP. To avoid model bias, the occupancies of
the Tyr203 side chain atoms and all chromophore atoms were set to
zero during the first several rounds of refinement. Constrained NCS
averaging over the A and B chains in the asymmetric unit was
applied, initial refinement was carried out to 3.5 .ANG. only, and
the electron density maps (2Fo-Fc and Fo-Fc) were averaged. These
maps were then inspected, and the model adjusted using O (Jones et
al., Acta Crystallogr. Sect. A 47:110, 1991), followed by
additional positional refinement to 2.5 .ANG.. Chromophore and
Tyr203 densities were very clear, and both were planar. The model
was edited to include these groups in refinement, and solvent
molecules were added where appropriate. B-factors were refined
using a strong correlation between neighboring atoms due to the
relatively low resolution.
[0174] Comparison of the Structure of YFP and YFP H148G
[0175] YFP crystallized in 2.2 M Na/K phosphate at pH 6.9 in
spacegroup P2.sub.12.sub.12, with 2 molecules per asymmetric unit
(chains A and B). The GFP S65T structure was used as a search model
for molecular replacement against a 3.0 .ANG. dataset using the
program AMoRe (Navaza, Acta Crystallogr. A50:157-63, 1994), and the
structure was refined. Later, the refined structure of YFP H148G
(see below) was used for phasing and refinement against a 2.5
.ANG.dataset. Even though the introduced Tyr203 and the chromophore
itself were not modeled during early cycles of refinement, clear
electron density for a planar chromophore and a stacked Tyr203
phenol was immediately apparent. Non-crystallographic symmetry
(NCS) constraints were employed throughout refinement of the model
using TNT, and maps were averaged. At the end of refinement,
non-averaged maps for the A-and B-chain in the asymmetric unit were
calculated and compared to each other. No obvious features were
identifiable that would suggest significant differences between the
two chains. A test run of refinement without any NCS constraints
confirmed that the differences would be smaller than the rms error
of a 2.5 .ANG. structure. Therefore, the NCS constraints were not
relaxed or eliminated. Data collection and atomic model statistics
are shown in Table 33. The final R-factor of the YFP model was
19.2% for all data between 20 and 2.5 .ANG. resolution.
33TABLE 33 Data collection and atomic model statistics of YFP and
YFP H148G. YFP YFP H148G Total observations 53,039 29,904 Unique
reflections 18,916 7,373 Completeness.sup.a 92% 99% Completeness
(shell.sup.b) 94% 97% Number of crystals 2 1 .sup.Rmerge.sup.c (%)
8.0% 6.5% Resolution 2.5 .ANG. 2.6 .ANG. Atomic model statistics:
Spacegroup P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 Molecules
per asymm, unit 2 1 Crystallographic R-factor 0.192 0.159 Protein
atoms 1,810 1,810 Solvent atoms per asymmm, unit 130 30 Bond length
deviations (.ANG.) 0.013 0.012 Bond angle deviations (.degree.)
1.76 2.07 Thermal parameter restraints (.ANG..sup.2) 4.53 3.82
.sup.aCompleteness is the ratio of the number of observed I > 0
divided by the theoretically possible number of intensities.
.sup.bShell is the highest resolution shell (2.56 to 2.50 .ANG. for
YFP, and 2.80 to 2.60 .ANG. for YFP H148G) .sup.cR.sub.merge =
.SIGMA. .vertline.I.sub.hk1 - <I>.vertline. /.SIGMA. < I
> where < I > = average of individual measurements of
I.sub.hk1.
[0176] The refined YFP structure clearly shows that the overall
fold is undisturbed, with an rms deviation from the GFP S65T
structure of 0.36 .ANG. for .alpha.-carbons. Three larger contact
areas with adjacent molecules were identified. The largest of these
covers about 722 .ANG.2 of one monomer surface, includes a series
of hydrophobic residues consisting of Ala206, Phe223, and Leu221,
and also a number of hydrophilic contacts. This interface is
essentially identical to the dimer interface for WT GFP described
by Yang et al., Nature Biotech. 14:1246-51, 1996. High salt
conditions during crystallization experiments appear to favor
dimerization, as has been suggested previously (Palm et al., Nat.
Struct. Biol. 4:361-65, 1997).
[0177] YFP H148G crystallized as a monomer in the presence of
polyethylene glycol and acetate at pH 4.6 in spacegroup
P2.sub.12.sub.12.sub.1, isomorphous to S65T (Ormo et al., Science
273:1392-95, 1996) and the blue-emission variant BFP (Wachter et
al., Biochemistry 36:9759-65, 1997). Molecular replacement using
the S65T structure for phasing and refinement gave a final model
with an R-factor of 15.9% for all data between 24.0 and 2.6 .ANG.
(Table 33). As with YFP, electron density for a stacked phenol was
clearly visible even before the Tyr203 ring was modeled. The rms
deviation between YFP H148G and S65T a-carbons is 0.31 .ANG., and
the deviation between YFP H148G and YFP a-carbons is 0.35 .ANG..
The b-strands of the two YFP variants overlay closely in all areas
except around the C.sub..alpha. of residue 148 where a movement of
1.1 .ANG. is observed. This movement has not been observed in other
pH 4.6 structures grown under similar conditions and crystallizing
in the same space group, such as the BFP structure (Wachter et al.,
Biochemistry 36:9759-65, 1997). Residue 148 and adjacent residues
are not involved in crystal contacts, further indicating that the
observed movement is due to the H148G substitution, not
crystallization conditions.
[0178] .pi.-Stacking of the Introduced Phenol
[0179] Electron densities of the chromophore and the phenol ring of
Tyr203 appeared to be completely planar before the atoms for these
groups were added to the model. When the tyrosine side chain was
first introduced into the model, it was modelled as co-planar to
the chromophore. Refinement consistently rotated the phenol ring by
12 with respect to the chromophore plane in both YFP and YFP H148G.
FIG. 9 shows the electron density of the refined YFP chromophore
structure together with the phenol ring of Tyr203. The distance of
the closest approach between atoms of the two interacting rings is
3.3 to 3.4 .ANG., and occurs at that edge of the chromophore plane
that is opposite the exo-methylene bond (FIG. 9). It appears that
the phenol tilts towards this area of the chromophore since it is
more open, with fewer atoms to clash with sterically.
[0180] The distance of largest separation between the rings is 3.5
to 3.8 .ANG., and occurs at the opposite edge, where steric clash
with the exo-methylene carbon could occur. This range of
plane-to-plane distances is typical for face-to-face .pi. to .pi.
stacking interactions found in proteins, and consistent with
interaction energy calculations that show a potential energy
minimum for two horizontally stacked benzene molecules with a
vertical separation of 3.3 .ANG. (Burley et al., J. Am. Chem. Soc.
108, 7995-8001, 1986). A recent analysis of protein structures has
led to the conclusion that aromatic ring interactions in an
off-centered parallel orientation have an energetically favorable,
stabilizing effect, and in fact are the preferred interactions
(McGaughey et al., J. Biol. Chem. 273, 15458-63, 1998).
[0181] Positional Shift of the Chromophore
[0182] The entire chromophore ring system of YFP has moved out
towards the protein surface by about 0.9 .ANG. when compared to
S65T or WT GFP. The chromophore of YFP H148G has Moved in the same
direction but to a lesser extent, about 0.5 .ANG.. Overlay of all
.alpha.-carbons shows that this shift is very much a local effect,
only involving residues 65 to 68. The overlay suggests that this
shift may be due to the compensating effects of the V68L and S65G
substitutions. The Leu68 C.delta.1 occupies the same space as the
original Val68 C.gamma.1, whereas the Leu68 backbone is displaced
so that the chromophore is pushed further out towards the protein
surface. As part of the same movement, the C.alpha. of Gly65 is
pushed into the position of the wild-type C.sub..beta. of Ser65.
The V68L and S65G substitutions had been previously found to
significantly increase the brightness of GFP-expressing cells
(Cormack et al., Gene 173:33, 1996) in a WT background, and were
suggested to either improve folding at 37.degree. or increase the
rate of chromophore formation. It is unclear at this point why the
chromophore is not shifted to the same extent in the YFP and YFP
H148G structures, though both of them incorporate the V68L and S65G
mutations.
[0183] Even though the imidazolinone ring of the YFPs is not in the
same position as in WT GFP (Brejc et al., Proc. Natl. Acad. Sci.
USA. 94:2306-11, 1997), S65T (Ormo et al. Science 273:1392-95,
1996), and blue-fluorescent protein BFP (Wachter et al.,
Biochemistry 36:9759-65, 1997), no electron density consistent with
partially formed or unformed chromophore is observed. This
indicates that the machinery to generate the chromophore is not
only intact, but more flexible than previously thought. Apparently,
the exact positions of the backbone atoms of residues 65 and 67
that undergo the cyclization reaction is not as crucial as was
previously suggested, based on the nearly exact superposition of
the imidazolinone rings observed in WT GFP, S65T, and BFP (Yang et
al., Nature Biotech. 14:1246-51, 1996; Brejc, K. et al., Proc.
Natl. Acad. Sci. USA 94:2306-2311, 1997; Ormo et al., Science
273:1392-95, 1996; Palm et al., Nat. Struct. Biol. 4:361-65, 1997;
Wachter et al., Biochemistry 36:9759-65, 1997).
[0184] Chromophore Spectral Properties, Charge State and Hydrogen
Bonding Interactions
[0185] The spectral properties of the YFPs were examined. Small
aliquots of protein (16 mg/ml) were diluted 48-fold into 75 mM
buffer (acetate, phosphate, Tris, or CHES), 140 mM NaCl, and then
scanned for absorbance between 250 and 600 nm (Shimadzu 2101
spectrophotometer at medium scan rate and room temperature). The
optical density at 514 or 512 nm was plotted as a function of pH
and computer-fitted to a Nitration curve (Kaleidagraph.TM.,
SynergySoftware).
[0186] Fluorescence measurements were carried out on a Hitachi
F4500 fluorescence spectrophotometer at a constant protein
concentration of approximately 0.01 mg/ml, with buffer conditions
identical to those of absorbance measurements. The excitation
wavelength was set to the absorbance maximum of the long-wave band
of the particular mutant. The emission was scanned between 500 and
600 nm, and peak emission intensity was plotted as a function of pH
and curve-fitted.
[0187] Like S65T (Kneen et al., Biophys. J. 74:1591-99, 1998), the
YFPs have two absorbance maxima whose relative ratio is
pH-dependent (FIG. 8 and Table 34). The UV absorption peaks at 392
(YFP) or 397 nm (YFP H148G) have been ascribed to the neutral
chromophore, whereas the visible absorption peaks at 514 (YFP) or
512 nm (YFP H148G) have been ascribed to the anionic chromophore
(Niwa et al., Proc. Natl. Acad. Sci. (USA) 93: 13617-22, 1996). The
lower energy peak exhibits clear vibrational structure as indicated
by the pronounced shoulder at 480-490 nm, and its mirror-image
relationship with the emission band is striking (FIG. 8). These
features are consistent with luminescence properties of large and
rigid systems in condensed phases (Barltrop et al., Principles of
Photochemistry, John Wiley and Sons, New York, 1978, pp. 51-52 and
78-79), and may be more pronounced in the YFPs due to decreased
chromophore flexibility in the presence of the stacked phenol. Both
YFPs fluoresce intensely when excited at the longer-wavelength
band, with maximum emission occurring at 528 nm (FIG. 8).
Fluorescence is extremely weak when the excitation occurs at the
shorter-wavelength band (Table 34), even if the experiment is
carried out at a pH where this peak dominates. The chromophore pKa
in the intact protein was determined to be 7.00(.+-.0.03) for YFP
and 8.02 (.+-.0.01) for YFP H148G by absorbance measurements at
varying pH. The pKa values determined by fluorescence were 6.95
(.+-.0.03) and 7.93 (.+-.0.04), respectively, for the two variants.
The YFP pKa is remarkably similar to that of
34TABLE 34 Summary of Absorption and Emission Maxima. absorbance
absorbance emission.sup.a emission.sup.a band #1 band #2 band band
WT GFP 398 475 460/598 504 S65T 394 489 (weak) 511 YFP 392 514
(weak) 528 HFP H148G 397 51122 (weak) 528 .sup.aThe emission band
#1 results from excitation at the absorbance peak #1, and the
emission band #2 results from excitation at the absorbance peak
#2.
[0188] EYFP (S65G/S72A/T203Y/H231L). All titration curves gave an
excellent fit to a single pKa value.
[0189] It is likely that the charge state of the chromophore is
mixed in the YFP crystals which were grown at pH 7, and which is
the chromophore pKa. In YFP, His148 is directly hydrogen-bonded to
the phenolic end of the chromophore. Its electron density is
well-defined, suggesting that the imidazole ring does not change
position when the chromophore ionizes. It is therefore unlikely
that structural rearrangements in the immediate chromophore
environment occur in response to changes in chromophore charge
state. In both the YFP and YFP H148G structures, the phenolic end
of the chromophore is nearly in H-bonding contact with bulk solvent
via two ordered waters, and therefore may not be as tightly
embedded in the protein as in WT and S65T (Brejc et al., Proc.
Natl. Acad. Sci. USA. 94:2306-11, 1997, Ormo et al., Science
273:1392-95, 1996]. Structural readjustments to accommodate the
anion may only affect solvent molecules.
[0190] The strong hydrogen bond to Arg96 that has been suggested to
play a role in the chemistry of backbone cyclization (Ormo et al.,
above) is maintained in both structures. The carbonyl oxygen of the
chromophore imidazolinone ring interacts with two hydrogen bond
donors, Arg96 and Gln69 in YFP, and Arg96 and Gln94 in YFP H148G.
This compares to similar interactions with Arg96 and Gln94 in WT
and S65T. The Glu222 carboxy oxygen approaches the chromophore
imidazolinone ring nitrogen to within 3.0 (YFP) and 3.3 .ANG. (YFP
H148G), considerably closer than in WT and S65T (4.3 and 4.0 .ANG.,
respectively). This close approach appears to be related to the
chromophore positional shift described above. Distance and geometry
for hydrogen bonding between Glu222 and the chromophore ring
nitrogen are excellent in YFP, and somewhat less optimal in YFP
H148G, where the presumed H-bond makes roughly a 45.degree. angle
with the chromophore plane. The YFP structure is the first GFP
structure solved that suggests H-bonding interactions of the
heterocyclic ring nitrogen originating from Tyr66. The most likely
interpretation in terms of charge states is a deprotonated ring
nitrogen and a protonated Glu222, rendering both groups neutral,
however, it is clear that they share a proton.
[0191] Solvent-Accessible Surface and Cavities
[0192] The mutation H148G was introduced into YFP to examine the
effects of solvent accessibility on the fluorescent properties and
the ionization constant of the chromophore. In all GFP structures
examined to date, the .beta.-barrel is somewhat perturbed around
the phenolic end of the chromophore. The .beta.-strand that covers
the chromophore in that area bulges out around His148, so that the
backbone from residue 144 to 150 is not directly hydrogen-bonded to
the adjacent backbone between residues 165 and 170. Rather, they
are laced together by forming H-bonds with the imidazole ring of
His148 (Arg168 backbone N to His148 N.sub..epsilon.2 in S65T and WT
GFP) and several water molecules. The phenolic end of the
chromophore is located directly "behind" the ring of His148. It was
anticipated that substitution of His with Gly would open up a
solvent channel to the chromophore in the absence of other
structural perturbances, or perhaps to permit the bulge to
close.
[0193] The crystal structure clearly shows this anticipated solvent
channel as an invagination of the protein surface with no ordered
solvent molecules within. Elimination of the imidazole ring in the
H148G substitution only leads to minor structural rearrangements of
protein groups. The .beta.-strands do not close up to form a
directly H-bonded sheet between residues 144 and 150. Instead, the
C.alpha. of residue 148 has actually moved in the opposite
direction by 1.1 .ANG., causing an even larger strand separation
between the backbones of residues 148 and 168. The side chain of
Ile167 has moved by 1.1 .ANG. towards the space previously occupied
by the imidazole ring. Nevertheless, direct solvent access to the
phenolic end of the chromophore is greatly improved. Calculation of
the solvent-accessible area of the chromophore using a probe sphere
of radius of 1.4 .ANG. (Connolly, Science 221:709-13, 1983), as
implemented by UCSF MidasPlus (UCSF MidasPlus.RTM., Computer
Graphics Laboratory, University of San Francisco, Calif. 94143),
shows that 22% of the chromophore surface is solvent-accessible
only the phenolic end of the chromophore is exposed to exterior
solvent, though, due to the opening in the protein wall. The
phenolic oxygen of the chromophore is also hydrogen-bonded to a
water molecule that is near H-bonding distance to a surface water,
though a 1.4 .ANG. probe cannot access the chromophore via this
path. If both the solvent channel as well as this hydrogen bond are
included, 8% of the chromophore surface is accessible to exterior
solvent, entirely at the phenolic end, and 14% is accessible to
interior solvent due to contact with internal cavities.
[0194] YFP H148G contains two larger interior cavities that are in
contact with the chromophore cavity and filled with some ordered
waters. The cavity that was largest in S65T has decreased in size
from approximately 127 .ANG.3 (S65T) to 88 .ANG.3 (YFP H148G),
because some of the space is now filled with the phenol of Tyr203.
In YFP, this cavity is not accessible to a 1.4 .ANG. probe at all
since several groups have moved into this space. The more
significant structural adjustments are C.sub..gamma.2 of Val224,
which has moved by 1.4 .ANG., and C.sub..delta.1 of Leu42 which has
moved by 2.0 .ANG., essentially filling the cavity. The second
larger cavity in contact with the chromophore is nearly invariant
for S65T, YFP, and YFP H148G, and is between 98 and 103 .ANG..sup.3
in size.
[0195] Solvent Accessibility to the Chromophore
[0196] YFP H148G was found to be highly fluorescent, with bright
greenish-yellow color under ordinary day light. The light-emitting
properties of the fluorophore do not appear to be changed to any
extent by the introduction of a solvent channel to the chromophore,
indicating that significant quenching does not occur.
[0197] Since the protein fold is entirely intact in YFP H148G in
spite of the generation of an opening in the .beta.-barrel, the
H148G substitution may be especially useful for allowing access of
various small-molecule species to the chromophore. This
substitution may be introduced into other GFP variants with a
larger cavity adjacent to the chromophore, such as S65T [7] or
S65G, allowing for analyte binding studies where specific spectral
shifts due to the interaction with small molecules or ions of
interest could be monitored. The highest ionization constant of all
variants examined to date is found for the YFP H148G mutant with a
pKa of 8.0. In this mutant, the chromophore is solvent exposed,
consistent with a similarly high pKa when the protein is denatured
(Nageswara et al., Biophys. J. 32:630-32, 1980).
Other Embodiments
[0198] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
38 1 716 DNA Aequorea victoria CDS (1)...(714) Green fluorescent
protein 1 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att
ctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
Leu Val 1 5 10 15 gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct
gtc agt gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt
acc ctt aaa ttt att tgc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
Thr Leu Lys Phe Ile Cys 35 40 45 act act gga aaa cta cct gtt cca
tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu Pro Val Pro
Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgc
ttt tca aga tac cca gat cat atg aaa cgg 240 Ser Tyr Gly Val Gln Cys
Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac ttt ttc
aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata
ttt ttc aaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110
aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att 384
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa ttg gaa tac
aac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
Asn 130 135 140 tat aac tca cac aat gta tac atc atg gca gac aaa caa
aag aat gga 480 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln
Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaa att aga cac aac
att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn
Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tat caa caa
aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr Gln Gln
Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aac
cat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac
gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct
gct ggg att aca cat ggc atg gat gaa cta tac aaa 714 Thr Ala Ala Gly
Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 ta 716 2 238
PRT Aequorea victoria 2 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His
Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr
Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90
95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala
Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile
Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215
220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230
235 3 239 PRT Aequorea victoria VARIANT (0)...(0) EGFP 3 Met Val
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20
25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr
Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met
Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150
155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr
Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 4 239 PRT Aequorea
victoria VARIANT (0)...(0) EYFP 4 Met Val Ser Lys Gly Glu Glu Leu
Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp
Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60
Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65
70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185
190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu
195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
Leu Tyr Lys 225 230 235 5 239 PRT Aequorea victoria VARIANT
(0)...(0) EYFP-V68L/Q69K 5 Met Val Ser Lys Gly Glu Glu Leu Phe Thr
Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn
Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly
Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85
90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile
Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe
Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val
Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210
215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235 6 239 PRT Aequorea victoria VARIANT (0)...(0) ECFP 6
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5
10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser
Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60 Leu Thr Trp Gly Val Gln Cys Phe Ser
Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser
Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile
Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135
140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu
Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr
Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys
Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 7 238 PRT
Aequorea victoria VARIANT (0)...(0) YFP H148G 7 Met Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40
45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe
50 55 60 Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met
Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser Gly
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170
175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala
Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp
Glu Leu Tyr Lys 225 230 235 8 238 PRT Aequorea victoria VARIANT
(0)...(0) YFP H148Q 8 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His
Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr
Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Gly Tyr Gly
Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90
95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser Gln Asn Val Tyr Ile Met Ala
Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile
Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu
Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser 195 200 205 Lys
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215
220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230
235 9 239 PRT Aequorea victoria VARIANT (0)...(0) EYFP-H148G 9 Met
Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10
15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg
Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140
Asn Tyr Asn Ser Gly Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145
150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp
Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu
Ser Tyr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg
Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile
Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 10 239 PRT Aequorea
victoria VARIANT (0)...(0) EYFP-H148Q 10 Met Val Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50
55 60 Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met
Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu
85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser Gln Asn Val Tyr
Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu
Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200
205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
Lys 225 230 235 11 720 DNA Aequorea victoria misc_feature (0)...(0)
EGFP 11 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc
accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta
cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact
tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca
tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac
aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac
ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca
cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
720 12 720 DNA Aequorea victoria misc_feature (0)...(0) EYFP 12
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc
tgcccgtgcc ctggcccacc 180 ctcgtgacca ccttcggcta cggcgtgcag
tgcttcgccc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc
cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg
acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc
gacggccccg tgctgctgcc cgacaaccac 600 tacctgagct accagtccgc
cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt
tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 13 720
DNA Aequorea victoria misc_feature (0)...(0) ECFP 13 atggtgagca
agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacag gttcagcgtg tccggcgagg gcgagggcga tgccacctac
120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc
gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc
gaaggctacg tccaggagcg taccatcttc 300 ttcaaggacg acggcaacta
caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac
480 ggcatcaagg cccacttcaa gatccgccac aacatcgagg acggcagcgt
gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg
tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa
gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc
cgccgggatc actctcggca tggacgagct gtacaagtaa 720 14 720 DNA Aequorea
victoria misc_feature (0)...(0) EYFP-V68L/Q69K 14 atggtgagca
agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180 ctcgtgacca ccttcggcta cggcctgaag tgcttcgccc
gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc
gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta
caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg
tgctgctgcc cgacaaccac 600 tacctgagct accagtccgc cctgagcaaa
gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc
cgccgggatc actctcggca tggacgagct gtacaagtaa 720 15 714 DNA Aequorea
victoria misc_feature (0)...(0) YFP H148G 15 atgagtaaag gagaagaact
tttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg
ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
180 gtcactactt tcggttatgg tcttcaatgc tttgcaagat acccagatca
tatgaaacgg 240 catgactttt tcaagagtgc catgcccgaa ggttatgttc
aggaaagaac tatatttttc 300 aaagatgacg ggaactacaa gacacgtgct
gaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaagg
tattgatttt aaagaagatg gaaacattct tggacacaaa 420 ttggaataca
actataactc aggcaatgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
540 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
caaccattac 600 ctgtcctatc aatctgccct ttcgaaagat cccaacgaaa
agagagacca catggtcctt 660 cttgagtttg taacagctgc tgggattaca
catggcatgg atgaactata caaa 714 16 714 DNA Aequorea victoria
misc_feature (0)...(0) YFP H148Q 16 atgagtaaag gagaagaact
tttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg
ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
180 gtcactactt tcggttatgg tcttcaatgc tttgcaagat acccagatca
tatgaaacgg 240 catgactttt tcaagagtgc catgcccgaa ggttatgttc
aggaaagaac tatatttttc 300 aaagatgacg ggaactacaa gacacgtgct
gaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaagg
tattgatttt aaagaagatg gaaacattct tggacacaaa 420 ttggaataca
actataactc aggcaatgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
540 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
caaccattac 600 ctgtcctatc aatctgccct ttcgaaagat cccaacgaaa
agagagacca catggtcctt 660 cttgagtttg taacagctgc tgggattaca
catggcatgg atgaactata caaa 714 17 720 DNA Aequorea victoria
misc_feature (0)...(0) EYFP-H148G 17 atggtgagca agggcgagga
gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa
acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
180 ctcgtgacca ccttcggcta cggcgtgcag tgcttcgccc gctaccccga
ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg
tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc
gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa
gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt
acaactacaa cagcggcaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
cgacaaccac 600 tacctgagct accagtccgc cctgagcaaa gaccccaacg
agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc
actctcggca tggacgagct gtacaagtaa 720 18 720 DNA Aequorea victoria
misc_feature (0)...(0) EYFP-H148Q 18 atggtgagca agggcgagga
gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa
acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
180 ctcgtgacca ccttcggcta cggcgtgcag tgcttcgccc gctaccccga
ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg
tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc
gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa
gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt
acaactacaa cagccagaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
cgacaaccac 600 tacctgagct accagtccgc cctgagcaaa gaccccaacg
agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc
actctcggca tggacgagct gtacaagtaa 720 19 255 PRT Aequorea victoria
VARIANT (0)...(0) mito-ECFP 19 Met Leu Ser Leu Arg Gln Ser Ile Arg
Phe Phe Lys Arg Ser Gly Ile 1 5 10 15 Met Val Ser Lys Gly Glu Glu
Leu Phe Thr Gly Val Val Pro Ile Leu 20 25 30 Val Glu Leu Asp Gly
Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 35 40 45 Glu Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 Cys
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70
75 80 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
Lys 85 90 95 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr Ile Ser
His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 165 170 175 Gly Ile
Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195
200 205 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu 210 215 220 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe 225 230 235 240 Val Thr Ala Ala Gly Ile Thr Leu Gly Met
Asp Glu Leu Tyr Lys 245 250 255 20 255 PRT Aequorea victoria
VARIANT (0)...(0) mito-EYFP 20 Met Leu Ser Leu Arg Gln Ser Ile Arg
Phe Phe Lys Arg Ser Gly Ile 1 5 10 15 Met Val Ser Lys Gly Glu Glu
Leu Phe Thr Gly Val Val Pro Ile Leu 20 25 30 Val Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 35 40 45 Glu Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 Cys
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70
75 80 Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met
Lys 85 90 95 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr Asn Ser
His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 165 170 175 Gly Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195
200 205 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala
Leu 210 215 220 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe 225 230 235 240 Val Thr Ala Ala Gly Ile Thr Leu Gly Met
Asp Glu Leu Tyr Lys 245 250 255 21 323 PRT Aequorea victoria
VARIANT (0)...(0) GT-EGFP 21 Met Arg Leu Arg Glu Pro Leu Leu Ser
Gly Ala Ala Met Pro Gly Ala 1 5 10 15 Ser Leu Gln Arg Ala Cys Arg
Leu Leu Val Ala Val Cys Ala Leu His 20 25 30 Leu Gly Val Thr Leu
Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser Arg 35 40 45 Leu Pro Gln
Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser Asn 50 55 60 Ser
Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly Gly 65 70
75 80 Ala Met Asp Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
Val 85 90 95 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
His Lys Phe 100 105 110 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
Tyr Gly Lys Leu Thr 115 120 125 Leu Lys Phe Ile Cys Thr Thr Gly Lys
Leu Pro Val Pro Trp Pro Thr 130 135 140 Leu Val Thr Thr Leu Thr Tyr
Gly Val Gln Cys Phe Ser Arg Tyr Pro 145 150 155 160 Asp His Met Lys
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 165 170 175 Tyr Val
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 180 185 190
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 195
200 205 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
His 210 215 220 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
Met Ala Asp 225 230 235 240 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
Lys Ile Arg His Asn Ile 245 250 255 Glu Asp Gly Ser Val Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro 260 265 270 Ile Gly Asp Gly Pro Val
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 275 280 285 Gln Ser Ala Leu
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 290 295 300 Leu Leu
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 305 310 315
320 Leu Tyr Lys 22 323 PRT Aequorea victoria VARIANT (0)...(0)
GT-EYFP 22 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ala Ala Met Pro
Gly Ala 1 5 10 15 Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val
Cys Ala Leu His 20 25 30 Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala
Gly Arg Asp Leu Ser Arg 35 40 45 Leu Pro Gln Leu Val Gly Val Ser
Thr Pro Leu Gln Gly Gly Ser Asn 50 55 60 Ser Ala Ala Ala Ile Gly
Gln Ser Ser Gly Glu Leu Arg Thr Gly Gly 65 70 75 80 Ala Met Asp Pro
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 85 90 95 Val Pro
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 100 105 110
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 115
120 125 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr 130 135 140 Leu Val Thr Thr Phe Gly Tyr Gly Val Gln Cys Phe Ala
Arg Tyr Pro 145 150 155 160 Asp His Met Lys Gln His Asp Phe Phe Lys
Ser Ala Met Pro Glu Gly 165 170 175 Tyr Val Gln Glu Arg Thr Ile Phe
Phe Lys Asp Asp Gly Asn Tyr Lys 180 185 190 Thr Arg Ala Glu Val Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 195 200 205 Glu Leu Lys Gly
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 210 215 220 Lys Leu
Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 225 230 235
240 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile
245 250 255 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
Thr Pro 260 265 270 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
Tyr Leu Ser Tyr 275 280 285 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
Lys Arg Asp His Met Val 290 295 300 Leu Leu Glu Phe Val Thr Ala Ala
Gly Ile Thr Leu Gly Met Asp Glu 305 310 315 320 Leu Tyr Lys 23 263
PRT Aequorea victoria VARIANT (0)...(0) mito-YFP H148G 23 Met Leu
Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15
Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr Ser Lys Gly Glu Glu Leu 20
25 30 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
Asn 35 40 45 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
Ala Thr Tyr 50 55 60 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
Gly Lys Leu Pro Val 65 70 75 80 Pro Trp Pro Thr Leu Val Thr Thr Phe
Gly Tyr Gly Leu Gln Cys Phe 85 90
95 Ala Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala
100 105 110 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
Asp Asp 115 120 125 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
Gly Asp Thr Leu 130 135 140 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
Phe Lys Glu Asp Gly Asn 145 150 155 160 Ile Leu Gly His Lys Leu Glu
Tyr Asn Tyr Asn Ser Gly Asn Val Tyr 165 170 175 Ile Met Ala Asp Lys
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 180 185 190 Arg His Asn
Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 195 200 205 Gln
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 210 215
220 Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg
225 230 235 240 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
Ile Thr His 245 250 255 Gly Met Asp Glu Leu Tyr Lys 260 24 263 PRT
Aequorea victoria VARIANT (0)...(0) mito-YFP H148Q 24 Met Leu Arg
Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe
Arg Asn Ile Leu Arg Leu Gln Ser Thr Ser Lys Gly Glu Glu Leu 20 25
30 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
35 40 45 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
Thr Tyr 50 55 60 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly
Lys Leu Pro Val 65 70 75 80 Pro Trp Pro Thr Leu Val Thr Thr Phe Gly
Tyr Gly Leu Gln Cys Phe 85 90 95 Ala Arg Tyr Pro Asp His Met Lys
Arg His Asp Phe Phe Lys Ser Ala 100 105 110 Met Pro Glu Gly Tyr Val
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 115 120 125 Gly Asn Tyr Lys
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 130 135 140 Val Asn
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 145 150 155
160 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser Gln Asn Val Tyr
165 170 175 Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
Lys Ile 180 185 190 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala
Asp His Tyr Gln 195 200 205 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
Leu Leu Pro Asp Asn His 210 215 220 Tyr Leu Ser Tyr Gln Ser Ala Leu
Ser Lys Asp Pro Asn Glu Lys Arg 225 230 235 240 Asp His Met Val Leu
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His 245 250 255 Gly Met Asp
Glu Leu Tyr Lys 260 25 265 PRT Aequorea victoria VARIANT (0)...(0)
mito-EYFP-H148G 25 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val
Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr
Met Val Ser Lys Gly Glu 20 25 30 Glu Leu Phe Thr Gly Val Val Pro
Ile Leu Val Glu Leu Asp Gly Asp 35 40 45 Val Asn Gly His Lys Phe
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 50 55 60 Thr Tyr Gly Lys
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 65 70 75 80 Pro Val
Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gln 85 90 95
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 100
105 110 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
Lys 115 120 125 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
Glu Gly Asp 130 135 140 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
Asp Phe Lys Glu Asp 145 150 155 160 Gly Asn Ile Leu Gly His Lys Leu
Glu Tyr Asn Tyr Asn Ser Gly Asn 165 170 175 Val Tyr Ile Met Ala Asp
Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 180 185 190 Lys Ile Arg His
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 195 200 205 Tyr Gln
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 210 215 220
Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 225
230 235 240 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
Gly Ile 245 250 255 Thr Leu Gly Met Asp Glu Leu Tyr Lys 260 265 26
265 PRT Aequorea victoria VARIANT (0)...(0) mito-EYFP-H148Q 26 Met
Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10
15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr Met Val Ser Lys Gly Glu
20 25 30 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
Gly Asp 35 40 45 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
Glu Gly Asp Ala 50 55 60 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
Cys Thr Thr Gly Lys Leu 65 70 75 80 Pro Val Pro Trp Pro Thr Leu Val
Thr Thr Phe Gly Tyr Gly Val Gln 85 90 95 Cys Phe Ala Arg Tyr Pro
Asp His Met Lys Gln His Asp Phe Phe Lys 100 105 110 Ser Ala Met Pro
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 115 120 125 Asp Asp
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 130 135 140
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 145
150 155 160 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser
Gln Asn 165 170 175 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
Lys Val Asn Phe 180 185 190 Lys Ile Arg His Asn Ile Glu Asp Gly Ser
Val Gln Leu Ala Asp His 195 200 205 Tyr Gln Gln Asn Thr Pro Ile Gly
Asp Gly Pro Val Leu Leu Pro Asp 210 215 220 Asn His Tyr Leu Ser Tyr
Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 225 230 235 240 Lys Arg Asp
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 245 250 255 Thr
Leu Gly Met Asp Glu Leu Tyr Lys 260 265 27 972 DNA Aequorea
victoria misc_feature (0)...(0) GT-ECFP 27 atgaggcttc gggagccgct
cctgagcggc gccgcgatgc caggcgcgtc cctacagcgg 60 gcctgccgcc
tgctcgtggc cgtctgcgct ctgcaccttg gcgtcaccct cgtttactac 120
ctggctggcc gcgacctgag ccgcctgccc caactggtcg gagtctccac accgctgcag
180 ggcggctcga acagtgccgc cgccatcggg cagtcctccg gggagctccg
gaccggaggg 240 gccatggatc ccatggtgag caagggcgag gagctgttca
ccggggtggt gcccatcctg 300 gtcgagctgg acggcgacgt aaacggccac
aggttcagcg tgtccggcga gggcgagggc 360 gatgccacct acggcaagct
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 420 ccctggccca
ccctcgtgac caccctgacc tggggcgtgc agtgcttcag ccgctacccc 480
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
540 cgtaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
gaagttcgag 600 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
acttcaagga ggacggcaac 660 atcctggggc acaagctgga gtacaactac
atcagccaca acgtctatat caccgccgac 720 aagcagaaga acggcatcaa
ggcccacttc aagatccgcc acaacatcga ggacggcagc 780 gtgcagctcg
ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 840
cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
900 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
catggacgag 960 ctgtacaagt aa 972 28 768 DNA Aequorea victoria
misc_feature (0)...(0) mito-EYFP 28 atgctgagcc tgcgccagag
catccgcttc ttcaagcgca gcggcatcat ggtgagcaag 60 ggcgaggagc
tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 120
ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
180 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
cgtgaccacc 240 ttcggctacg gcgtgcagtg cttcgcccgc taccccgacc
acatgaagca gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtc
caggagcgca ccatcttctt caaggacgac 360 ggcaactaca agacccgcgc
cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 420 gagctgaagg
gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 480
aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
540 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
ccactaccag 600 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
acaaccacta cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgag
aagcgcgatc acatggtcct gctggagttc 720 gtgaccgccg ccgggatcac
tctcggcatg gacgagctgt acaagtaa 768 29 972 DNA Aequorea victoria
misc_feature (0)...(0) GT-EGFP 29 atgaggcttc gggagccgct cctgagcggc
gccgcgatgc caggcgcgtc cctacagcgg 60 gcctgccgcc tgctcgtggc
cgtctgcgct ctgcaccttg gcgtcaccct cgtttactac 120 ctggctggcc
gcgacctgag ccgcctgccc caactggtcg gagtctccac accgctgcag 180
ggcggctcga acagtgccgc cgccatcggg cagtcctccg gggagctccg gaccggaggg
240 gccatggatc ccatggtgag caagggcgag gagctgttca ccggggtggt
gcccatcctg 300 gtcgagctgg acggcgacgt aaacggccac aagttcagcg
tgtccggcga gggcgagggc 360 gatgccacct acggcaagct gaccctgaag
ttcatctgca ccaccggcaa gctgcccgtg 420 ccctggccca ccctcgtgac
caccctgacc tacggcgtgc agtgcttcag ccgctacccc 480 gaccacatga
agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 540
cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
600 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
ggacggcaac 660 atcctggggc acaagctgga gtacaactac aacagccaca
acgtctatat catggccgac 720 aagcagaaga acggcatcaa ggtgaacttc
aagatccgcc acaacatcga ggacggcagc 780 gtgcagctcg ccgaccacta
ccagcagaac acccccatcg gcgacggccc cgtgctgctg 840 cccgacaacc
actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 900
gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
960 ctgtacaagt aa 972 30 972 DNA Aequorea victoria misc_feature
(0)...(0) GT-EYFP 30 atgaggcttc gggagccgct cctgagcggc gccgcgatgc
caggcgcgtc cctacagcgg 60 gcctgccgcc tgctcgtggc cgtctgcgct
ctgcaccttg gcgtcaccct cgtttactac 120 ctggctggcc gcgacctgag
ccgcctgccc caactggtcg gagtctccac accgctgcag 180 ggcggctcga
acagtgccgc cgccatcggg cagtcctccg gggagctccg gaccggaggg 240
gccatggatc ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
300 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
gggcgagggc 360 gatgccacct acggcaagct gaccctgaag ttcatctgca
ccaccggcaa gctgcccgtg 420 ccctggccca ccctcgtgac caccttcggc
tacggcgtgc agtgcttcgc ccgctacccc 480 gaccacatga agcagcacga
cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 540 cgcaccatct
tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 600
ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
660 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
catggccgac 720 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
acaacatcga ggacggcagc 780 gtgcagctcg ccgaccacta ccagcagaac
acccccatcg gcgacggccc cgtgctgctg 840 cccgacaacc actacctgag
ctaccagtcc gccctgagca aagaccccaa cgagaagcgc 900 gatcacatgg
tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 960
ctgtacaagt aa 972 31 762 DNA Aequorea victoria misc_feature
(0)...(0) mito-YFP H148G 31 atgctgagcc tgcgccagag catccgcttc
ttcaagcgca gcggcatcat gagtaaagga 60 gaagaacttt tcactggagt
tgtcccaatt cttgttgaat tagatggtga tgttaatggg 120 cacaaatttt
ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 180
aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc
240 ggttatggtc ttcaatgctt tgcaagatac ccagatcata tgaaacggca
tgactttttc 300 aagagtgcca tgcccgaagg ttatgttcag gaaagaacta
tatttttcaa agatgacggg 360 aactacaaga cacgtgctga agtcaagttt
gaaggtgata cccttgttaa tagaatcgag 420 ttaaaaggta ttgattttaa
agaagatgga aacattcttg gacacaaatt ggaatacaac 480 tataactcag
gcaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac 540
ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
600 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
gtcctatcaa 660 tctgcccttt cgaaagatcc caacgaaaag agagaccaca
tggtccttct tgagtttgta 720 acagctgctg ggattacaca tggcatggat
gaactataca aa 762 32 762 DNA Aequorea victoria misc_feature
(0)...(0) mito-YFP H148Q 32 atgctgagcc tgcgccagag catccgcttc
ttcaagcgca gcggcatcat gagtaaagga 60 gaagaacttt tcactggagt
tgtcccaatt cttgttgaat tagatggtga tgttaatggg 120 cacaaatttt
ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 180
aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc
240 ggttatggtc ttcaatgctt tgcaagatac ccagatcata tgaaacggca
tgactttttc 300 aagagtgcca tgcccgaagg ttatgttcag gaaagaacta
tatttttcaa agatgacggg 360 aactacaaga cacgtgctga agtcaagttt
gaaggtgata cccttgttaa tagaatcgag 420 ttaaaaggta ttgattttaa
agaagatgga aacattcttg gacacaaatt ggaatacaac 480 tataactcag
gcaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac 540
ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
600 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
gtcctatcaa 660 tctgcccttt cgaaagatcc caacgaaaag agagaccaca
tggtccttct tgagtttgta 720 acagctgctg ggattacaca tggcatggat
gaactataca aa 762 33 768 DNA Aequorea victoria misc_feature
(0)...(0) mito-EYFP-H148G 33 atgctgagcc tgcgccagag catccgcttc
ttcaagcgca gcggcatcat ggtgagcaag 60 ggcgaggagc tgttcaccgg
ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 120 ggccacaagt
tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 180
ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
240 ttcggctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca
gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtc caggagcgca
ccatcttctt caaggacgac 360 ggcaactaca agacccgcgc cgaggtgaag
ttcgagggcg acaccctggt gaaccgcatc 420 gagctgaagg gcatcgactt
caaggaggac ggcaacatcc tggggcacaa gctggagtac 480 aactacaaca
gcggcaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 540
aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
600 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
acatggtcct gctggagttc 720 gtgaccgccg ccgggatcac tctcggcatg
gacgagctgt acaagtaa 768 34 768 DNA Aequorea victoria misc_feature
(0)...(0) mito-EYFP-H148Q 34 atgctgagcc tgcgccagag catccgcttc
ttcaagcgca gcggcatcat ggtgagcaag 60 ggcgaggagc tgttcaccgg
ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 120 ggccacaagt
tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 180
ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
240 ttcggctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca
gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtc caggagcgca
ccatcttctt caaggacgac 360 ggcaactaca agacccgcgc cgaggtgaag
ttcgagggcg acaccctggt gaaccgcatc 420 gagctgaagg gcatcgactt
caaggaggac ggcaacatcc tggggcacaa gctggagtac 480 aactacaaca
gccagaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 540
aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
600 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
acatggtcct gctggagttc 720 gtgaccgccg ccgggatcac tctcggcatg
gacgagctgt acaagtaa 768 35 5 PRT Homo sapiens 35 Lys Lys Lys Arg
Lys 1 5 36 26 PRT Homo sapiens 36 Met Leu Arg Thr Ser Ser Leu Phe
Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg
Leu Gln Ser Thr 20 25 37 4 PRT Homo sapiens 37 Lys Asp Glu Leu 1 38
4 PRT Aequorea victoria VARIANT (0)...(0) Linker sequence 38 Arg
Ser Gly Ile 1
* * * * *