U.S. patent application number 14/251535 was filed with the patent office on 2014-08-07 for optical control of protein activity and localization by fusion to photochromic protein domains.
This patent application is currently assigned to Office of Technology Licensing. The applicant listed for this patent is Michael Lin, Xin Zhou. Invention is credited to Michael Lin, Xin Zhou.
Application Number | 20140220615 14/251535 |
Document ID | / |
Family ID | 49212191 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220615 |
Kind Code |
A1 |
Zhou; Xin ; et al. |
August 7, 2014 |
OPTICAL CONTROL OF PROTEIN ACTIVITY AND LOCALIZATION BY FUSION TO
PHOTOCHROMIC PROTEIN DOMAINS
Abstract
Engineered fusion proteins comprising photochromic protein
domains are disclosed. In particular, the inventors have
constructed fusion proteins containing photoswitchable photochromic
fluorescent protein domains linked to selected proteins and shown
that such fusion proteins can be used to control the activity or
localization of selected proteins with light.
Inventors: |
Zhou; Xin; (Stanford,
CA) ; Lin; Michael; (Stanford, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhou; Xin
Lin; Michael |
Stanford
Stanford |
CA
CA |
US
US |
|
|
Assignee: |
Office of Technology
Licensing
Palo Alto
CA
|
Family ID: |
49212191 |
Appl. No.: |
14/251535 |
Filed: |
April 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13848346 |
Mar 21, 2013 |
8735096 |
|
|
14251535 |
|
|
|
|
61614492 |
Mar 22, 2012 |
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Current U.S.
Class: |
435/29 ;
435/173.1 |
Current CPC
Class: |
C07K 14/001 20130101;
C12N 13/00 20130101; C12Q 1/02 20130101 |
Class at
Publication: |
435/29 ;
435/173.1 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. A method for controlling the activity of a selected polypeptide
of interest with light, the method comprising: a) preparing a
fusion protein comprising at least two photochromic polypeptides
connected to a selected polypeptide of interest, wherein a first
photochromic polypeptide is connected to the N-terminus of the
selected polypeptide of interest and a second photochromic
polypeptide is connected to the C-terminus of the selected
polypeptide of interest, wherein the first photochromic polypeptide
and the second photochromic polypeptide are capable of associating
with each other, wherein the oligomerization state of the first
photochromic polypeptide and the second photochromic polypeptide is
controllable with light, wherein at least one photochromic
polypeptide is an rsTagRFP polypeptide or an mApple polypeptide;
and b) illuminating the fusion protein with light at a wavelength
that induces intramolecular dimerization of the first photochromic
polypeptide and the second photochromic polypeptide, such that the
activity of the selected polypeptide of interest is
inactivated.
2. The method of claim 1, further comprising illuminating the
fusion protein with light at a wavelength that induces dissociation
of the first photochromic polypeptide from the second photochromic
polypeptide, such that the activity of the selected polypeptide is
restored.
3. The method of claim 1, further comprising visualizing the
localization of the selected polypeptide by detecting fluorescence
of the fusion protein resulting from the dimerization of the first
photochromic polypeptide and the second photochromic
polypeptide.
4. The method of claim 1, further comprising detecting inactivation
of the selected polypeptide by measuring fluorescence from
dimerization of the first photochromic polypeptide and the second
photochromic polypeptide.
5. The method of claim 1, further comprising detecting inactivation
of the selected polypeptide by measuring the activity of the
selected polypeptide.
6. The method of claim 1, wherein fluorescence of the fusion
protein is detected by a fluorimeter, a fluorescence microscope, a
fluorescence microplate reader, a fluorometric imaging plate
reader, or fluorescence-activated cell sorting.
7. The method of claim 1, wherein the first photochromic
polypeptide or the second photochromic polypeptide comprises: a) an
amino acid sequence selected from the group consisting of SEQ ID
NO:7 and SEQ ID NO:9; or b) an amino acid sequence having at least
95% identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:7 and SEQ ID NO:9, wherein the polypeptide
has fluorescence and oligomerization characteristics.
8. A method for controlling the localization of a selected
polypeptide of interest with light, the method comprising: a)
preparing a first fusion protein comprising a photochromic
polypeptide connected to a targeting sequence; b) preparing a
second fusion protein comprising a photochromic polypeptide
connected to the selected polypeptide of interest; c) introducing
the first fusion protein and the second fusion protein into a cell,
wherein the localization sequence targets the first fusion protein
to a particular subcellular location; d) illuminating the fusion
proteins with light at a wavelength that induces oligomerization of
the photochromic polypeptide in the first fusion protein with the
photochromic polypeptide in the second fusion protein, such that
the selected polypeptide accumulates at the subcellular
location.
9. The method of claim 8, further comprising illuminating the
fusion proteins with light at a wavelength that induces
dissociation of the photochromic polypeptides, such that the
selected polypeptide in the second fusion protein is released from
the subcellular location.
10. The method of claim 8, further comprising visualizing the
localization of the selected polypeptide by detecting fluorescence
of the fusion proteins resulting from the oligomerization of the
photochromic polypeptides.
11. The method of claim 8, wherein the targeting sequence is
selected from the group consisting of a secretory protein signal
sequence, a membrane protein signal sequence, a nuclear
localization sequence, a nucleolar localization signal sequence, an
endoplasmic reticulum localization sequence, a peroxisome
localization sequence, a mitochondrial localization sequence, and a
protein binding motif sequence.
12. The method of claim 8, wherein fluorescence of the fusion
proteins are detected by a fluorometer, a fluorescence microscope,
a fluorescence microplate reader, a fluorometric imaging plate
reader, or fluorescence-activated cell sorting.
13. The method of claim 8, wherein the photochromic polypeptide in
the first fusion protein and the photochromic polypeptide in the
second fusion protein are selected from the group consisting of a
Dronpa polypeptide, a Padron polypeptide, an rsTagRFP polypeptide,
and an mApple polypeptide.
14. The method of claim 13, wherein the photochromic polypeptide of
the first fusion protein or the second fusion protein comprises: a)
an amino acid sequence selected from the group consisting of SEQ ID
NOS:1, 3, 5, 7, and 9; or b) an amino acid sequence having at least
95% identity to an amino acid sequence selected from the group
consisting of SEQ ID NOS:1, 3, 5, 7, and 9.
15. The method of claim 13, wherein the photochromic polypeptide in
the first fusion protein is a Dronpa-145N polypeptide or a
Padron-145N polypeptide.
16. The method of claim 13, wherein the photochromic polypeptide in
the first fusion protein is a Dronpa-145K polypeptide.
17. The method of claim 13, wherein the photochromic polypeptide in
the second fusion protein is a Dronpa-145N polypeptide or a
Padron-145N polypeptide.
18. The method of claim 13, wherein the photochromic polypeptide in
the second fusion protein is a Dronpa-145K polypeptide.
19. A method for controlling the localization of a selected
polypeptide of interest with light, the method comprising: a)
preparing a fusion protein comprising a photochromic polypeptide, a
targeting sequence, and the selected polypeptide of interest; b)
introducing the fusion protein into a cell, wherein the
localization sequence targets the fusion protein to a particular
subcellular location; c) illuminating the fusion protein with light
at a wavelength that induces oligomerization of the photochromic
polypeptide in the fusion protein with photochromic polypeptides in
other fusion proteins of the same type, said fusion proteins
comprising the selected polypeptide of interest, such that the
selected polypeptide of interest accumulates at the subcellular
location.
20. The method of claim 19, further comprising illuminating the
fusion protein with light at a wavelength that induces dissociation
of the photochromic polypeptides, such that the selected
polypeptide of interest in the fusion protein is released from the
subcellular location.
21. The method of claim 19, further comprising visualizing the
localization of the selected polypeptide of interest by detecting
fluorescence of the fusion protein resulting from the
oligomerization with the photochromic polypeptides of the other
fusion proteins.
22. The method of claim 19, wherein the targeting sequence is
selected from the group consisting of a secretory protein signal
sequence, a membrane protein signal sequence, a nuclear
localization sequence, a nucleolar localization signal sequence, an
endoplasmic reticulum localization sequence, a peroxisome
localization sequence, a mitochondrial localization sequence, and a
protein binding motif sequence.
23. The method of claim 19, wherein the photochromic polypeptide of
the fusion protein comprises an amino acid sequence selected from
the group consisting of a Dronpa polypeptide, a Padron polypeptide,
an rsTagRFP polypeptide, and an mApple polypeptide.
24. The method of claim 23, wherein the photochromic polypeptide of
the fusion protein comprises: a) an amino acid sequence selected
from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9; or b) an
amino acid sequence having at least 95% identity to an amino acid
sequence selected from the group consisting of SEQ ID NOS:1, 3, 5,
7, and 9, wherein the photochromic polypeptide has fluorescence and
oligomerization characteristics.
25. The method of claim 23, wherein the photochromic polypeptide of
the fusion protein is a Dronpa-145N polypeptide or a Padron-145N
polypeptide.
26. The method of claim 23, wherein the photochromic polypeptide of
the fusion protein is an mApple-162H-164A polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S.
application Ser. No. 13/848,346, filed Mar. 21, 2013, which claims
benefit benefit under 35 U.S.C. .sctn.119(e) of provisional
application Ser. No. 61/614,492, filed Mar. 22, 2012, all of which
applications are hereby incorporated by reference in their
entireties.
TECHNICAL FIELD
[0002] The present invention pertains generally to the field of
protein engineering and methods of controlling the activity or
cellular localization of proteins. In particular, the invention
relates to engineered fusion proteins comprising photochromic
protein domains and methods of using them to control protein
activity or localization with light.
BACKGROUND
[0003] The ability to control protein localization and activity
would be enormously beneficial for understanding and modulating
protein function in physiological processes. Several approaches
have been developed previously for optical control of protein
activity using natural proteins and protein domains that change
conformation upon light absorption, for example, using proteins
such as rhodopsins, phytochromes, and cryptochromes, and LOV
domains from phototropins and FKF1 (Airan et al. (2009) Nature
458:1025-1029; Inoue et al. (2005) Nat. Methods 2:415-418; Kennedy
et al. (2010) Nat. Methods 7:973-975; Levskaya et al. (2009) Nature
461:997-1001; Szobota et al. (2007) Neuron 54:535-545; Wu et al.
(2009) Nature 461:104-108; and Yazawa et al. (2009) Nat.
Biotechnol. 27:941-945). However, widespread implementation of
these methods has been hindered by various problems, including the
limited applicability of the methods to only specific signaling
pathways (Airan et al., supra), the need for exogenous cofactors
(Levskaya et al., supra), slow kinetics of induction (Yazawa et
al., supra), undesirable light-independent dimerization (Kennedy et
al., supra), or the toxicity of light at blue wavelengths (Szobota
et al., supra; Wu et al., supra; Yazawa et al., supra).
Furthermore, of all these strategies, only fusion to LOV domains
has been used to control the activity of a single protein, but this
method generally requires extensive customization (Wu et al.,
supra; Strickland et al. (2008) Proc. Natl. Acad. Sci. U.S.A.
105:10709-10714; Strickland et al. (2010) Nat. Methods 7:623-626;
and Wu et al. (2011) Methods Enzymol. 497:393-407). In addition,
none of these light-absorbing domains are capable of controlling
both protein localization by intermolecular interactions and
function of a single polypeptide chain.
[0004] Thus, there remains a need for a simple to use system for
controlling protein localization and activity with light, which can
be readily applied to a wide range of proteins.
SUMMARY
[0005] The invention relates to engineered fusion proteins
comprising photochromic protein domains. In particular, the
inventors have constructed fusion proteins containing a
photoswitchable photochromic fluorescent protein. The inventors
have further shown that fusion proteins comprising one or more
photochromic fluorescent protein domains linked to a selected
protein of interest can be used to control the activity or
localization of the selected protein using light.
[0006] In one aspect, the invention includes a fusion protein
comprising at least one photochromic polypeptide connected to a
selected polypeptide of interest, wherein the oligomerization state
of the photochromic polypeptide is controllable with light. The
photochromic polypeptide may be a photochromic protein, or a
variant or polypeptide fragment thereof having fluorescence
characteristics, wherein the fluorescence characteristics of the
fusion protein are dependent on the oligomerization state of the
photochromic polypeptide. For example, photochromic proteins
including, but not limited to Dronpa, Padron, rsTagRFP, and mApple,
or a variant or polypeptide fragment thereof having fluorescence
characteristics (e.g., Dronpa-145N, Padron-145N, or
mApple-162H-164A), may be used in fusion constructs. In certain
embodiments, the fusion protein comprises at least one photochromic
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NOS:1, 3, 5, 7, and 9 or a variant
thereof comprising a sequence having at least about 80-100%
sequence identity thereto, including any percent identity within
this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.
[0007] In certain embodiments, the fusion protein comprises at
least two photochromic polypeptides, wherein a first photochromic
polypeptide is connected to the N-terminus of the selected
polypeptide of interest and a second photochromic polypeptide is
connected to the C-terminus of the selected polypeptide of
interest, wherein the oligomerization state of the first
photochromic polypeptide and the second photochromic polypeptide is
controllable with light. For example, the fusion protein may
comprise two or more Dronpa, Padron, rsTagRFP, or mApple
polypeptides. The photochromic polypeptides in the fusion protein
can be the same or different.
[0008] In certain embodiments, the fusion protein comprises a
Dronpa protein, or a variant or polypeptide fragment thereof having
fluorescence and oligomerization characteristics. The fusion
protein may comprise at least one Dronpa 145N or Dronpa 145 K
polypeptide. In certain embodiments, the fusion protein comprises
two Dronpa polypeptides, which can be the same or different. For
example, the fusion protein may comprise two Dronpa 145N
polypeptides, or two Dronpa 145K polypeptides, or a Dronpa 145K
polypeptide and a Dronpa 145N polypeptide.
[0009] In certain embodiments, the fusion protein further comprises
one or more linkers connecting polypeptides within the fusion
protein. Linkers are typically short peptide sequences of 2-30
amino acid residues, often composed of glycine and/or serine
residues. Linker sequences that can be used in the practice of the
invention include, but are not limited to [Gly].sub.x,
[Gly-Ser].sub.x, [Gly-Gly-Ser-Gly].sub.x, [Ser-Ala-Gly-Gly].sub.x,
and [Gly-Gly-Gly-Gly-Ser].sub.x, wherein x=1-15, and GSAT, SEG, and
Z-EGFR linkers.
[0010] In certain embodiments, the fusion protein further comprises
a targeting sequence. Targeting sequences that can be used in the
practice of the invention include, but are not limited to a
secretory protein signal sequence, a membrane protein signal
sequence, a nuclear localization sequence, a nucleolar localization
signal sequence, an endoplasmic reticulum localization sequence, a
peroxisome localization sequence, a mitochondrial localization
sequence, and a protein-protein interaction motif sequence.
[0011] In certain embodiments, the fusion protein further comprises
a tag. Tags that can be used in the practice of the invention
include, but are not limited to a His-tag, a Strep-tag, a TAP-tag,
an S-tag, an SBP-tag, an Arg-tag, a calmodulin-binding peptide tag,
a cellulose-binding domain tag, a DsbA tag, a c-myc tag, a
glutathione S-transferase tag, a FLAG tag, a HAT-tag, a
maltose-binding protein tag, a NusA tag, and a thioredoxin tag.
[0012] The fusion proteins described herein can be used to control
the activity or localization of a selected protein, which may be a
membrane protein, a receptor, a hormone, a transport protein, a
transcription factor, a cytoskeletal protein, an extracellular
matrix protein, a signal-transduction protein, an enzyme, or any
other protein of interest. The fusion protein may comprise the
entire protein, or a biologically active domain (e.g., a catalytic
domain, a ligand binding domain, or a protein-protein interaction
domain), or a polypeptide fragment of the selected protein of
interest.
[0013] In another aspect, the invention includes a method for
controlling the activity of a selected polypeptide of interest with
light. The method comprises (i) preparing a fusion protein
comprising a first photochromic polypeptide connected to the
N-terminus of the selected polypeptide of interest and a second
photochromic polypeptide connected to the C-terminus of the
selected polypeptide of interest; (ii) illuminating the fusion
protein with light at a wavelength that induces intramolecular
dimerization of the first photochromic polypeptide and the second
photochromic polypeptide (e.g., about 405 nm for some fusions with
Dronpa 145N or 145K), such that the activity of the selected
polypeptide of interest is inactivated. In certain embodiments, the
method further comprises illuminating the fusion protein with light
at a wavelength that induces dissociation of the first photochromic
polypeptide from the second photochromic polypeptide (e.g., about
480-500 nm for some fusions with Dronpa 145N or 145K), such that
the activity of the selected polypeptide is restored. Localization
of the selected polypeptide as well as inactivation of the selected
polypeptide can be visualized by detecting fluorescence of the
fusion protein resulting from intramolecular dimerization of the
first photochromic polypeptide and the second photochromic
polypeptide in the fusion protein. Inactivation of the selected
polypeptide can further be assessed by measuring the activity of
the selected polypeptide.
[0014] In another aspect, the invention includes a method for
controlling the localization of a selected polypeptide of interest
with light. The method comprises (i) preparing a first fusion
protein comprising a photochromic polypeptide connected to a
targeting sequence; (ii) preparing a second fusion protein
comprising a photochromic polypeptide connected to the selected
polypeptide of interest; (iii) introducing the first fusion protein
and the second fusion protein into a cell, wherein the localization
sequence targets the first fusion protein to a particular
subcellular location; (iv) and illuminating the fusion proteins
with light at a wavelength that induces oligomerization of the
photochromic polypeptide in the first fusion protein with the
photochromic polypeptide in the second fusion protein (e.g., about
405 nm for some fusions with Dronpa 145N or 145K), such that the
selected polypeptide of interest accumulates at the subcellular
location. In certain embodiments, the method further comprises
illuminating the fusion proteins with light at a wavelength that
induces dissociation of the photochromic polypeptides (e.g., about
480-500 nm for some fusions with Dronpa 145N or 145K), such that
the selected polypeptide in the second fusion protein is released
from the subcellular location. Localization of the selected
polypeptide can be visualized by detecting fluorescence of the
fusion proteins resulting from the oligomerization of the
photochromic polypeptides.
[0015] In another aspect, the invention includes a method for
controlling the localization of a selected polypeptide of interest
with light. The method comprises: (i) preparing a fusion protein
comprising a photochromic polypeptide, a targeting sequence, and
the selected polypeptide of interest; (ii) introducing the fusion
protein into a cell, wherein the localization sequence targets the
fusion protein to a particular subcellular location; and (iii)
illuminating the fusion protein with light at a wavelength that
induces oligomerization of the photochromic polypeptide in the
fusion protein with photochromic polypeptides in other fusion
proteins (e.g., 405 nm for some fusions with Dronpa 145N or 145K),
the other fusion proteins comprising the selected polypeptide, such
that the selected polypeptide accumulates at the subcellular
location. In certain embodiments, the method further comprises
illuminating the fusion protein with light at a wavelength that
induces dissociation of the photochromic polypeptides (e.g, about
480-500 nm for some fusions with Dronpa 145N or 145K), such that
the selected polypeptide in the fusion protein is released from the
subcellular location. Localization of the selected polypeptide can
be visualized by detecting fluorescence of the fusion protein
resulting from the oligomerization with photochromic polypeptides
of the other fusion proteins.
[0016] In another aspect, the invention includes a polynucleotide
encoding a fusion protein described herein. In one embodiment, the
polynucleotide is a recombinant polynucleotide comprising a
polynucleotide encoding a fusion protein operably linked to a
promoter. In certain embodiments, the recombinant polynucleotide
comprises a polynucleotide selected from the group consisting of: a
polynucleotide encoding a polypeptide comprising a sequence
selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9;
a polynucleotide encoding a polypeptide comprising a sequence
having at least 95% identity to a sequence selected from the group
consisting of SEQ ID NOS:1, 3, 5, 7, and 9; a polynucleotide
comprising a sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, and 10; and a polynucleotide comprising a sequence
having at least 95% identity to a sequence selected from the group
consisting of SEQ ID NOS:2, 4, 6, 8, and 10.
[0017] In another aspect, the invention includes a host cell
comprising a recombinant polynucleotide encoding a fusion protein
operably linked to a promoter.
[0018] In another aspect, the invention includes a method for
producing a fusion protein, the method comprising: transforming a
host cell with a recombinant polynucleotide encoding a fusion
protein operably linked to a promoter; culturing the transformed
host cell under conditions whereby the fusion protein is expressed;
and isolating the fusion protein from the host cell.
[0019] In another aspect, the invention includes a kit for
preparing or using fusion proteins according to the methods
described herein. Such kits may comprise one or more photochromic
polypeptides or fusion proteins, or nucleic acids encoding such
polypeptides or fusion proteins, or expression vectors, or cells,
or other reagents for preparing polypeptides and fusion proteins,
as described herein.
[0020] In the practice of the invention, the fluorescence of fusion
proteins can be monitored by any suitable method. For example,
fluorescence of fusion proteins can be detected by a fluorometer, a
fluorescence microscope, a fluorescence microplate reader, a
fluorometric imaging plate reader, or fluorescence-activated cell
sorting.
[0021] These and other embodiments of the subject invention will
readily occur to those of skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A-1F show the control of photochromic fluorescent
protein (FP) domain association by light. FIG. 1A shows a schematic
representation of the hypothesized bidirectional control of the
Dronpa145N oligomerization state by 500-nm cyan and 400-nm violet
light. FIG. 1B shows native polyacrylamide gel electrophoresis
(PAGE) of Dronpa145N (100 .mu.M), which demonstrated that 500 nm
light induced dissociation and 400 nm light induced
retetramerization. The mRuby2 (Lam et al. (2012) Nat. Methods
9:1005-1012), tdTomato, and dsRed2 (20 .mu.M) served as monomeric,
dimeric, and tetrameric standards, respectively. All proteins were
polyhistidine-tagged at the amino terminus (NT). FIG. 1C shows
absorbance spectra confirming that photoswitching is reversible.
FIG. 1D shows a schematic representation of the hypothesized
bidirectional conformational switching by light in a
Dronpa145K-Dronpa145N (K-N) tandem dimer. FIG. 1E shows that native
PAGE of the K-N tandem dimer demonstrated faster migration by the
K-N tandem dimer (100 .mu.M) after exposure to 500-nm light, an
effect that was reversed by 400-nm light. The asterisk marks the
location expected for tandem dimer migration, similar to tdTomato.
Some cleavage of the tandem dimer to a monomer in this protein
preparation was apparent. FIG. 1F shows absorbance spectra of K-N
tandem dimers confirming that photoswitching is reversible.
[0023] FIGS. 2A-2H show the control of photochromic FP domain
association by light in cells. FIG. 2A shows the experimental plan
for light-regulated interaction between Dronpa145N-CAAX (N-CAAX)
and mNeptune-Dronpa145N (mNeptune-N). FIG. 2B shows quantitation of
membrane Dronpa fluorescence during 490/20-nm illumination. FIG. 2C
shows that 490/20-nm light induced off-photoswitching of Dronpa and
loss of mNeptune from the plasma membrane (scale bar, 20 .mu.m).
FIG. 2D shows an intensity profile for the region between the
arrows shown in FIG. 2C. FIG. 2E shows the experimental plan for
light-regulated interaction between Dronpa145K-CAAX (K-CAAX) and
mNeptune-N. FIG. 2F shows the quantitation of membrane Dronpa
fluorescence during 490/20-nm illumination. FIG. 2G shows that
490/20-nm light induced off-photoswitching of Dronpa and loss of
mNeptune from the membrane. mNeptune reappeared at the membrane
after 3-seconds of on-photoswitching with 390/15-nm light (scale
bar, 20 .mu.m). FIG. 2H shows intensity profiles for the region
between the arrows shown in FIG. 2G.
[0024] FIGS. 3A-3H show a light-inducible single-chain guanine
nucleotide exchange factor (GEF). FIG. 3A shows the proposed
mechanism for photo-uncaging of N-I-N-CAAX activity (construct
contained Dronpa145N at the N-terminus of the intersectin (ITSN)
Dbl homology (DH) domain and Dronpa145N at the C-terminus followed
by the CAAX sequence). FIG. 3B shows off-photoswitching of Dronpa
fluorescence in N-I-N-CAAX versus 490/20-nm light dosage during
microscopy. Whole-cell fluorescence results from five cells were
quantified and normalized to the initial value. Error bars
represent standard deviation (SD). FIG. 3C shows that in NIH 3T3
cells expressing N-I-N-CAAX, 490/20-nm illumination for 30 seconds
(off-switching) followed by incubation at 37.degree. C. for 30
minutes resulted in robust induction of filopodia, as revealed by
mNeptune-Fascin. FIG. 3D shows that local illumination by 490/20-nm
light locally induced filopodia, marked by mNeptune-fascin, in NIH
3T3 cells expressing N-I-N-CAAX. The dotted curves indicate the
area of illumination. FIG. 3E shows the proposed mechanism for
photo-uncaging of K-I-N-CAAX activity (construct contained
Dronpa145K at the N-terminus of the intersectin (ITSN) Dbl homology
(DH) domain and Dronpa145N at the C-terminus followed by the CAAX
sequence). FIG. 3F shows off-photoswitching of Dronpa fluorescence
in K-I-N-CAAX versus 490/20-nm light dosage during microscopy. The
experiment was performed as described for FIG. 3B. FIG. 3G shows
that in NIH 3T3 cells expressing K-I-N-CAAX, exposure to 490/20-nm
light for 30 seconds (off-switching) followed by incubation at
37.degree. C. for 30 minutes resulted in robust induction of
filopodia. FIG. 3H shows that local illumination by 490/20-nm light
locally induced filopodia, marked by mNeptune-fascin, in NIH 3T3
cells expressing K-I-N-CAAX. The dotted curves indicate the area of
illumination. The scale bars in FIGS. 3C, 3D, 3G, and 3H are 20
.mu.m.
[0025] FIGS. 4A-4C show results with a light-inducible single-chain
protease, N-protease-N (Dronpa145N-protease-Dronpa145N fusion).
FIG. 4A shows the strategy for sensing activity of the N-protease-N
protein with mCherry-substrate-CAAX. FIG. 4B shows the distribution
of mCherry in cells expressing mCherry-substrateCAAX in the absence
(left) or presence (middle) of cotransfected K-protease. The chart
at right shows the fluorescence intensity profile along the line
between the arrows in the images. FIG. 4C shows that as expected
from its size (81 kD), N-protease-N was excluded from the nucleus
(left). Exposure to 490/20-nm light for 15 seconds induced
off-photoswitching of Dronpa fluorescence (Dronpa channel) and
induced release of mCherry from the membrane (mCherry channel). The
chart at right shows the intensity profile along the line between
the arrows in the images, which confirmed that mCherry fluorescence
decreases from the membrane and increases in the cytosol and
nucleus after illumination. The scale bars in FIGS. 4B and 4C are
20 .mu.m.
[0026] FIGS. 5A-5C show quantification of reversible photoswitching
of recombinant Dronpa constructs by fluorescence in vitro. FIG. 5A
shows a native PAGE, which demonstrated that Dronpa145K is purely
monomeric and 145N is predominantly tetrameric at concentrations
from 10 .mu.M to 100 .mu.M. The mRuby2, tdTomato, and dsRed2 served
as monomeric, dimeric, and tetrameric standards, respectively. FIG.
5B shows that fluorescence of Dronpa145N was switched off by 500 nm
light and switched back on by 400 nm light using the same
conditions as described for FIG. 1. Fluorescence was measured in
quadruplicate at 480/5 nm excitation and 530/5 nm emission and
intensities normalized to the initial value. Error bars represent
standard deviation. FIG. 5C shows that fluorescence of the
Dronpa145K-Dronpa145N tandem dimer was switched off by 500 nm light
and switched back on by 400 nm light using the same conditions as
described for FIG. 1.
[0027] FIGS. 6A and 6B show the directional specificity of membrane
recruitment of Dronpa. FIG. 6A shows that N-CAAX did not recruit
mNeptune-K to the membrane, possibly due to N--N intramembrane
homotetramerization outcompeting K-N heterodimerization. FIG. 6B
shows that K-CAAX was unable to recruit mNeptune-K to the membrane,
confirming mNeptune membrane localization required Dronpa
multimerization. Scale bars are 10 .mu.m.
[0028] FIGS. 7A-7D show protein caging by fusion to interacting FP
domains. FIG. 7A shows structural models of a DH domain caged by
two flanking Dronpa145N domains (top), or flanking Dronpa145K and
Dronpa145N domains (bottom). FIG. 7B shows the organization of the
control intersectin Dbl-homology domain (ITSN DH) constructs, caged
ITSN DH constructs, and a mNeptune-fascin filopodia reporter. FIG.
7C shows representative NIH 3T3 fibroblasts expressing K-CAAX,
K-I-CAAX, I-K-CAAX, N-I-N-CAAX, or K-I-N-CAAX (Dronpa channel) with
filopodia and lamellipodia marked by mNeptune-fascin (mNeptune
channel). The scale bar is 10 .mu.m. FIG. 7D shows the frequency of
filopodia or lamellipodia formation in cells transiently
transfected with various constructs. Cells showing lamellipodia or
more than one filopodium at one polygonal side were scored as
positive. Numbers above the bars are the number of cells in each
condition. All imaged cells were scored. The scale bars are 10
.mu.m.
[0029] FIGS. 8A-8D show the quantitation of filopodia and
lamellipodia production by Dronpa-intersectin fusion constructs at
different expression levels. FIG. 8A shows the distribution of
Dronpa fluorescence intensities from 37 cells transfected with
N-I-N-CAAX. Boundaries for defining low, medium, and high
expressers are shown as dotted lines. FIG. 8B shows the occurrence
of filopodia or lamellipodia is low in cells expressing low levels
of N-I-N-CAAX. FIG. 8C shows the distribution of Dronpa
fluorescence intensities from 50 cells transfected with K-I-N-CAAX.
Boundaries for defining low, medium, and high expressers are shown
as dotted lines. FIG. 8D shows that the occurrence of filopodia or
lamellipodia is low in cells expressing low levels of
K-I-N-CAAX.
[0030] FIGS. 9A-9D show that filopodia induction required both
light and a caged ITSN DH protein. FIG. 9A shows that cells
expressing N-I-N-CAAX did not produce filopodia without
illumination. Initial Dronpa was not imaged to avoid uncaging by
the 500 nm excitation light. FIG. 9B shows that cells expressing
K-I-N-CAAX did not produce filopodia under identical conditions
without illumination. Initial Dronpa was not imaged to avoid
uncaging by the 490/20 nm excitation light. FIG. 9C shows that in
cells expressing K-CAAX (lacking ITSN DH), 490/20 nm illumination
for 30 seconds (Dronpa off-switching) followed by incubation at
37.degree. C. for 30 minutes did not produce filopodia or
lamellipodia. FIG. 9D shows that quantitation of new filopodia and
lamellipodia formation with light stimulation in cells expressing
N-I-N-CAAX and K-I-N-CAAX. Numbers above the bars are the number of
cells in each condition. All imaged cells were scored. The scale
bars are 10 .mu.m.
[0031] FIG. 10 shows temporal regulation of filopodia by light
induction of intersectin activity. In a NIH3T3 cell expressing
N-I-N-CAAX, local uncaging with 500 nm light (frame 1, dotted
circle) induced local filopodia formation in 10 minutes (arrow,
frames 2-3). The cell was then globally illuminated with 400 nm
light to recage N-I-N-CAAX, then local uncaging was performed in a
new location with 500 nm light (frame 4, dotted circle). Filopodia
in the first uncaging location subsequently retracted (asterisk)
while new filopodia formed in the second uncaging location (arrows,
frames 5-7). The scale bar is 20 .mu.m.
[0032] FIG. 11 shows that optical induction of intersectin reveals
a role for Cdc42 in filopodia elongation. Uncaging of K-I-N-CAAX in
a cell expressing preexisting filopodia (arrows) results in
lengthening of the filopodia. New filopodia formation can also be
observed (asterisk). The scale bar is 20 .mu.m.
[0033] FIGS. 12A and 12B show that release of mCherry from the
mCherry-substrate-CAAX fusion required both light and the caged
protease. FIG. 12A shows that HEK293 cells expressing
mCherry-substrate-CAAX together with N-protease-N did not release
mCherry from the membrane in the absence of light stimulation. FIG.
12B shows that cells expressing mCherry-substrate-CAAX alone did
not release mCherry from the membrane even after light stimulation.
Scale bars are 10 .mu.m.
DETAILED DESCRIPTION
[0034] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of pharmacology,
chemistry, biochemistry, recombinant DNA techniques and immunology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Handbook of Experimental Immunology,
Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell
Scientific Publications); A. L. Lehninger, Biochemistry (Worth
Publishers, Inc., current addition); Sambrook, et al., Molecular
Cloning: A Laboratory Manual (3.sup.rd Edition, 2001); Methods In
Enzymology (S. Colowick and N. Kaplan eds., Academic Press,
Inc.).
[0035] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entireties.
I. DEFINITIONS
[0036] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0037] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a fusion protein" includes a
mixture of two or more fusion proteins, and the like.
[0038] The term "about," particularly in reference to a given
quantity, is meant to encompass deviations of plus or minus five
percent.
[0039] "Fluorescent protein" refers to any protein capable of
emitting light when excited with appropriate electromagnetic
radiation. Fluorescent proteins include proteins having amino acid
sequences that are either natural or engineered (e.g., Dronpa,
Padron, rsTagRFP, and mApple, and variants and derivatives
thereof).
[0040] A Dronpa polynucleotide, nucleic acid, oligonucleotide,
protein, polypeptide, or peptide refers to a molecule derived from
a coral of the genus Pectiniidae. The molecule need not be
physically derived from Pectiniidae, but may be synthetically or
recombinantly produced. A number of Dronpa nucleic acid and protein
sequences are known. Representative Dronpa sequences are presented
in SEQ ID NOS:1-4. Additional representative sequences are listed
in the National Center for Biotechnology Information (NCBI)
database. See, for example, NCBI entries: Accession Nos. AB180726,
ADE48854, BAD72874.1, 2IOV_D, 2IOV_C, 2IOV_B, 2IOV_A, 2POX_D,
2POX_C, 2POX_B, 2POX_A, AED56657, AED56658, AED56659, and AED56660;
all of which sequences (as entered by the date of filing of this
application) are herein incorporated by reference. Any of these
sequences or a variant thereof comprising a sequence having at
least about 80-100% sequence identity thereto, including any
percent identity within this range, such as 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence
identity thereto, can be used to construct a fusion protein, as
described herein.
[0041] A Padron polynucleotide, nucleic acid, oligonucleotide,
protein, polypeptide, or peptide refers to a molecule derived from
Echinophyllia sp. SC22. The molecule need not be physically derived
from Echinophyllia sp., but may be synthetically or recombinantly
produced. A number of Padron nucleic acid and protein sequences are
known. Representative Padron sequences are presented in SEQ ID NO:5
and SEQ ID NO:6. Additional representative sequences are listed in
the National Center for Biotechnology Information (NCBI) database.
See, for example, NCBI entries: Accession Nos. ACL36360, ACL98050,
EU983551, FJ014613, 3ZUL_A, 3ZUL_B, 3ZUL_C, 3ZUL_D, 3ZUL_E, 3ZUL_F,
3ZUJ_A, 3ZUJ_B, 3ZUJ_C, 3ZUJ_D, 3ZUJ_E, 3ZUJ_F, 3ZUF_A, 3ZUF_B,
3ZUF_C, 3ZUF_D, 3ZUF_E, and 3ZUF_F; all of which sequences (as
entered by the date of filing of this application) are herein
incorporated by reference. Any of these sequences or a variant
thereof comprising a sequence having at least about 80-100%
sequence identity thereto, including any percent identity within
this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be
used to construct a fusion protein, as described herein.
[0042] An rsTagRFP polynucleotide, nucleic acid, oligonucleotide,
protein, polypeptide, or peptide refers to a molecule derived from
Entacmaea quadricolor. The molecule need not be physically derived
from Entacmaea quadricolor, but may be synthetically or
recombinantly produced. A number of rsTagRFP nucleic acid and
protein sequences are known. Representative rsTagRFP sequences are
presented in SEQ ID NO:7 and SEQ ID NO:8. Additional representative
sequences are listed in the National Center for Biotechnology
Information (NCBI) database. See, for example, NCBI entries:
Accession Nos. 3U8C_A, 3U8C_B, 3U8C_C, 3U8C_D, 3U8A_A, 3U8A_B,
3U8A_C, 3U8A_D; all of which sequences (as entered by the date of
filing of this application) are herein incorporated by reference.
Any of these sequences or a variant thereof comprising a sequence
having at least about 80-100% sequence identity thereto, including
any percent identity within this range, such as 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence
identity thereto, can be used to construct a fusion protein, as
described herein.
[0043] An mApple polynucleotide, nucleic acid, oligonucleotide,
protein, polypeptide, or peptide refers to a molecule derived from
Discosoma sp. The molecule need not be physically derived from
Discosoma sp., but may be synthetically or recombinantly produced.
A number of mApple nucleic acid and protein sequences are known.
Representative mApple sequences are presented in SEQ ID NO:9 and
SEQ ID NO:10. Additional representative sequences are listed in the
National Center for Biotechnology Information (NCBI) database. See,
for example, NCBI entries: Accession Nos. ABC66097, DQ336160; all
of which sequences (as entered by the date of filing of this
application) are herein incorporated by reference. Any of these
sequences or a variant thereof comprising a sequence having at
least about 80-100% sequence identity thereto, including any
percent identity within this range, such as 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence
identity thereto, can be used to construct a fusion protein, as
described herein.
[0044] The terms "fusion protein," "fusion polypeptide," or
"photochromic fusion protein" as used herein refer to a fusion
comprising at least one photochromic polypeptide in combination
with a selected polypeptide of interest as part of a single
continuous chain of amino acids, which chain does not occur in
nature. The photochromic polypeptides and other selected
polypeptides may be connected directly to each other by peptide
bonds or may be separated by intervening amino acid sequences. The
fusion may include entire proteins or fragments thereof, including,
for example, sequences of Dronpa, Padron, rsTagRFP, mApple, or
variants thereof having fluorescence characteristics (e.g.,
Dronpa-145K, Dronpa-145N, Padron-145N, and mApple-162H-164A). The
fusion polypeptides may also contain sequences exogenous to the
photochromic or other selected polypeptides. For example, the
fusion may include targeting or localization sequences, tag
sequences, sequences of other fluorescent proteins (e.g., other
proteins with fluorescence characteristics that differ from Dronpa,
Padron, rsTagRFP, or mApple), or other chromophores. Moreover, the
fusion may contain sequences from multiple photochromic proteins,
or variants thereof, and/or other selected proteins. For example,
the fusion protein may comprise two or more Dronpa, Padron,
rsTagRFP, or mApple polypeptides, which can be the same or
different (e.g., two or more Dronpa 145K or Dronpa 145N
polypeptides, or a Dronpa 145K polypeptide and a Dronpa 145N
polypeptide simultaneously in the same fusion). Alternatively, the
fusion protein may comprise only one photochromic polypeptide,
which can be a wild-type polypeptide, or variant thereof.
[0045] The term "fluorescence characteristics" means an ability to
emit fluorescence by irradiation of excitation light. The
fluorescence characteristics of a fluorescent fusion protein
comprising a photochromic polypeptide or a variant thereof may be
comparable to or different from those of the fluorescent proteins
which have the amino acid sequences shown in SEQ ID NOS:1, 3, 5, 7,
and 9. Examples of parameters of the fluorescence characteristics
include fluorescence intensity, excitation wavelength, fluorescence
wavelength, and pH sensitivity.
[0046] The terms "polypeptide" and "protein" refer to a polymer of
amino acid residues and are not limited to a minimum length. Thus,
peptides, oligopeptides, dimers, multimers, and the like, are
included within the definition. Both full length proteins and
fragments thereof are encompassed by the definition. The terms also
include postexpression modifications of the polypeptide, for
example, glycosylation, acetylation, phosphorylation,
hydroxylation, and the like. Furthermore, for purposes of the
present invention, a "polypeptide" refers to a protein which
includes modifications, such as deletions, additions and
substitutions to the native sequence, so long as the protein
maintains the desired activity. These modifications may be
deliberate, as through site directed mutagenesis, or may be
accidental, such as through mutations of hosts which produce the
proteins or errors due to PCR amplification.
[0047] By "derivative" is intended any suitable modification of the
native polypeptide of interest, of a fragment of the native
polypeptide, or of their respective analogs, such as glycosylation,
phosphorylation, polymer conjugation (such as with polyethylene
glycol), or other addition of foreign moieties, as long as the
desired biological activity of the native polypeptide is retained.
Methods for making polypeptide fragments, analogs, and derivatives
are generally available in the art.
[0048] By "fragment" is intended a molecule consisting of only a
part of the intact full length sequence and structure. The fragment
can include a C-terminal deletion an N-terminal deletion, and/or an
internal deletion of the polypeptide. Active fragments of a
particular protein or polypeptide will generally include at least
about 5-10 contiguous amino acid residues of the full length
molecule, preferably at least about 15-25 contiguous amino acid
residues of the full length molecule, and most preferably at least
about 20-50 or more contiguous amino acid residues of the full
length molecule, or any integer between 5 amino acids and the full
length sequence, provided that the fragment in question retains
biological activity, such as catalytic activity, ligand binding
activity, regulatory activity, fluorescence or oligomerization
characteristics, as defined herein.
[0049] "Substantially purified" generally refers to isolation of a
substance (compound, polynucleotide, protein, polypeptide,
polypeptide composition) such that the substance comprises the
majority percent of the sample in which it resides. Typically in a
sample, a substantially purified component comprises 50%,
preferably 80%-85%, more preferably 90-95% of the sample.
Techniques for purifying polynucleotides and polypeptides of
interest are well-known in the art and include, for example,
ion-exchange chromatography, affinity chromatography and
sedimentation according to density.
[0050] By "isolated" is meant, when referring to a polypeptide,
that the indicated molecule is separate and discrete from the whole
organism with which the molecule is found in nature or is present
in the substantial absence of other biological macro molecules of
the same type. The term "isolated" with respect to a polynucleotide
is a nucleic acid molecule devoid, in whole or part, of sequences
normally associated with it in nature; or a sequence, as it exists
in nature, but having heterologous sequences in association
therewith; or a molecule disassociated from the chromosome.
[0051] As used herein, the terms "label" and "detectable label"
refer to a molecule capable of detection, including, but not
limited to, radioactive isotopes, fluorescers, chemiluminescers,
enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,
chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin
or haptens) and the like. The term "fluorescer" refers to a
substance or a portion thereof which is capable of exhibiting
fluorescence in the detectable range. The term also includes
fluorescent proteins and polypeptides.
[0052] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two nucleic acid, or
two polypeptide sequences are "substantially homologous" to each
other when the sequences exhibit at least about 50% sequence
identity, preferably at least about 75% sequence identity, more
preferably at least about 80% 85% sequence identity, more
preferably at least about 90% sequence identity, and most
preferably at least about 95% 98% sequence identity over a defined
length of the molecules. As used herein, substantially homologous
also refers to sequences showing complete identity to the specified
sequence.
[0053] In general, "identity" refers to an exact nucleotide to
nucleotide or amino acid to amino acid correspondence of two
polynucleotides or polypeptide sequences, respectively. Percent
identity can be determined by a direct comparison of the sequence
information between two molecules by aligning the sequences,
counting the exact number of matches between the two aligned
sequences, dividing by the length of the shorter sequence, and
multiplying the result by 100. Readily available computer programs
can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O.
in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5
Suppl. 3:353 358, National biomedical Research Foundation,
Washington, D.C., which adapts the local homology algorithm of
Smith and Waterman Advances in Appl. Math. 2:482 489, 1981 for
peptide analysis. Programs for determining nucleotide sequence
identity are available in the Wisconsin Sequence Analysis Package,
Version 8 (available from Genetics Computer Group, Madison, Wis.)
for example, the BESTFIT, FASTA and GAP programs, which also rely
on the Smith and Waterman algorithm. These programs are readily
utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package referred to above. For example, percent identity of a
particular nucleotide sequence to a reference sequence can be
determined using the homology algorithm of Smith and Waterman with
a default scoring table and a gap penalty of six nucleotide
positions.
[0054] Another method of establishing percent identity in the
context of the present invention is to use the MPSRCH package of
programs copyrighted by the University of Edinburgh, developed by
John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of
packages the Smith Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six).
From the data generated the "Match" value reflects "sequence
identity." Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non
redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR. Details of these programs are readily
available.
[0055] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with single
stranded specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0056] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, viral,
semisynthetic, or synthetic origin which, by virtue of its origin
or manipulation, is not associated with all or a portion of the
polynucleotide with which it is associated in nature. The term
"recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide. In general, the gene of interest is cloned and then
expressed in transformed organisms, as described further below. The
host organism expresses the foreign gene to produce the protein
under expression conditions.
[0057] The term "transformation" refers to the insertion of an
exogenous polynucleotide into a host cell, irrespective of the
method used for the insertion. For example, direct uptake,
transduction or f-mating are included. The exogenous polynucleotide
may be maintained as a non-integrated vector, for example, a
plasmid, or alternatively, may be integrated into the host
genome.
[0058] "Recombinant host cells", "host cells," "cells", "cell
lines," "cell cultures," and other such terms denoting
microorganisms or higher eukaryotic cell lines cultured as
unicellular entities refer to cells which can be, or have been,
used as recipients for recombinant vector or other transferred DNA,
and include the original progeny of the original cell which has
been transfected.
[0059] A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences (or "control elements"). The boundaries of the
coding sequence can be determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxy)
terminus. A coding sequence can include, but is not limited to,
cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA
sequences from viral or prokaryotic DNA, and even synthetic DNA
sequences. A transcription termination sequence may be located 3'
to the coding sequence.
[0060] Typical "control elements," include, but are not limited to,
transcription promoters, transcription enhancer elements,
transcription termination signals, polyadenylation sequences
(located 3' to the translation stop codon), sequences for
optimization of initiation of translation (located 5' to the coding
sequence), and translation termination sequences.
[0061] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, a given promoter operably linked to a
coding sequence is capable of effecting the expression of the
coding sequence when the proper enzymes are present. The promoter
need not be contiguous with the coding sequence, so long as it
functions to direct the expression thereof. Thus, for example,
intervening untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0062] "Encoded by" refers to a nucleic acid sequence which codes
for a polypeptide sequence, wherein the polypeptide sequence or a
portion thereof contains an amino acid sequence of at least 3 to 5
amino acids, more preferably at least 8 to 10 amino acids, and even
more preferably at least 15 to 20 amino acids from a polypeptide
encoded by the nucleic acid sequence.
[0063] "Expression cassette" or "expression construct" refers to an
assembly which is capable of directing the expression of the
sequence(s) or gene(s) of interest. An expression cassette
generally includes control elements, as described above, such as a
promoter which is operably linked to (so as to direct transcription
of) the sequence(s) or gene(s) of interest, and often includes a
polyadenylation sequence as well. Within certain embodiments of the
invention, the expression cassette described herein may be
contained within a plasmid construct. In addition to the components
of the expression cassette, the plasmid construct may also include,
one or more selectable markers, a signal which allows the plasmid
construct to exist as single stranded DNA (e.g., a M13 origin of
replication), at least one multiple cloning site, and a "mammalian"
origin of replication (e.g., a SV40 or adenovirus origin of
replication).
[0064] "Purified polynucleotide" refers to a polynucleotide of
interest or fragment thereof which is essentially free, e.g.,
contains less than about 50%, preferably less than about 70%, and
more preferably less than about at least 90%, of the protein with
which the polynucleotide is naturally associated. Techniques for
purifying polynucleotides of interest are well-known in the art and
include, for example, disruption of the cell containing the
polynucleotide with a chaotropic agent and separation of the
polynucleotide(s) and proteins by ion-exchange chromatography,
affinity chromatography and sedimentation according to density.
[0065] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001)
Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring
Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in
Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981)
Gene 13:197. Such techniques can be used to introduce one or more
exogenous DNA moieties into suitable host cells. The term refers to
both stable and transient uptake of the genetic material, and
includes uptake of peptide- or antibody-linked DNAs.
[0066] A "vector" is capable of transferring nucleic acid sequences
to target cells (e.g., viral vectors, non-viral vectors,
particulate carriers, and liposomes). Typically, "vector
construct," "expression vector," and "gene transfer vector," mean
any nucleic acid construct capable of directing the expression of a
nucleic acid of interest and which can transfer nucleic acid
sequences to target cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
[0067] The terms "variant," "analog" and "mutein" refer to
biologically active derivatives of the reference molecule that
retain desired activity, such as fluorescence or oligomerization
characteristics. In general, the terms "variant" and "analog" refer
to compounds having a native polypeptide sequence and structure
with one or more amino acid additions, substitutions (generally
conservative in nature) and/or deletions, relative to the native
molecule, so long as the modifications do not destroy biological
activity and which are "substantially homologous" to the reference
molecule as defined below. In general, the amino acid sequences of
such analogs will have a high degree of sequence homology to the
reference sequence, e.g., amino acid sequence homology of more than
50%, generally more than 60%-70%, even more particularly 80%-85% or
more, such as at least 90%-95% or more, when the two sequences are
aligned. Often, the analogs will include the same number of amino
acids but will include substitutions, as explained herein. The term
"mutein" further includes polypeptides having one or more amino
acid-like molecules including but not limited to compounds
comprising only amino and/or imino molecules, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring (e.g.,
synthetic), cyclized, branched molecules and the like. The term
also includes molecules comprising one or more N-substituted
glycine residues (a "peptoid") and other synthetic amino acids or
peptides. (See, e.g., U.S. Pat. Nos. 5,831,005; 5,877,278; and
5,977,301; Nguyen et al., Chem. Biol. (2000) 7:463-473; and Simon
et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for
descriptions of peptoids). Methods for making polypeptide analogs
and muteins are known in the art and are described further
below.
[0068] As explained above, analogs generally include substitutions
that are conservative in nature, i.e., those substitutions that
take place within a family of amino acids that are related in their
side chains. Specifically, amino acids are generally divided into
four families: (1) acidic--aspartate and glutamate; (2)
basic--lysine, arginine, histidine; (3) non-polar--alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cysteine, serine threonine, and tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. For example, it is reasonably predictable that an isolated
replacement of leucine with isoleucine or valine, an aspartate with
a glutamate, a threonine with a serine, or a similar conservative
replacement of an amino acid with a structurally related amino
acid, will not have a major effect on the biological activity. For
example, the polypeptide of interest may include up to about 5-10
conservative or non-conservative amino acid substitutions, or even
up to about 15-25 conservative or non-conservative amino acid
substitutions, or any integer between 5-25, so long as the desired
function of the molecule remains intact. One of skill in the art
may readily determine regions of the molecule of interest that can
tolerate change by reference to Hopp/Woods and Kyte-Doolittle
plots, well known in the art.
[0069] "Gene transfer" or "gene delivery" refers to methods or
systems for reliably inserting DNA or RNA of interest into a host
cell. Such methods can result in transient expression of
non-integrated transferred DNA, extrachromosomal replication and
expression of transferred replicons (e.g., episomes), or
integration of transferred genetic material into the genomic DNA of
host cells. Gene delivery expression vectors include, but are not
limited to, vectors derived from bacterial plasmid vectors, viral
vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia
viruses.
[0070] The term "derived from" is used herein to identify the
original source of a molecule but is not meant to limit the method
by which the molecule is made which can be, for example, by
chemical synthesis or recombinant means.
[0071] A polynucleotide "derived from" a designated sequence refers
to a polynucleotide sequence which comprises a contiguous sequence
of approximately at least about 6 nucleotides, preferably at least
about 8 nucleotides, more preferably at least about 10-12
nucleotides, and even more preferably at least about 15-20
nucleotides corresponding, i.e., identical or complementary to, a
region of the designated nucleotide sequence. The derived
polynucleotide will not necessarily be derived physically from the
nucleotide sequence of interest, but may be generated in any
manner, including, but not limited to, chemical synthesis,
replication, reverse transcription or transcription, which is based
on the information provided by the sequence of bases in the
region(s) from which the polynucleotide is derived. As such, it may
represent either a sense or an antisense orientation of the
original polynucleotide.
II. MODES OF CARRYING OUT THE INVENTION
[0072] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0073] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0074] The present invention is based on the discovery of
engineered fusion proteins comprising photochromic fluorescent
protein domains that can be used to control the activity or
localization of a selected protein of interest. In particular, the
inventors have constructed fusion proteins containing
photoswitchable variants of the fluorescent protein Dronpa (see
Example 1). Dronpa undergoes light-inducible oligomerization, which
converts Dronpa from a dark form to a bright form with detectable
fluorescence. Thus, fusions of Dronpa with a selected protein allow
the protein to be detected when Dronpa is converted to its bright
form. The inventors have further shown that fusion proteins
comprising Dronpa linked to a selected protein of interest can be
used to control the activity or localization of the selected
protein with light (see Example 1). In order to further an
understanding of the invention, a more detailed discussion is
provided below regarding photochromic fusion proteins and methods
of using them to control the activity and localization of
proteins.
[0075] A. Fusion Proteins
[0076] Fusion proteins comprise at least one photochromic
polypeptide connected to a selected polypeptide of interest. The
fusion protein can be designed to block or induce activity of the
selected polypeptide of interest, control its interactions with
other macromolecules, or direct its subcellular localization. The
polypeptide of interest selected for study may be from a membrane
protein, a receptor, a hormone, a transport protein, a
transcription factor, a cytoskeletal protein, an extracellular
matrix protein, a signal-transduction protein, an enzyme, or any
other protein of interest. The fusion protein may include entire
photochromic proteins, or biologically active domains or
polypeptide fragments, or variants thereof having fluorescence
characteristics (e.g., Dronpa-145K, Dronpa-145N, Padron-145N,
rsTagRFP, and mApple-162H-164A). In addition, the fusion protein
may comprise an entire selected protein of interest, or a
biologically active domain (e.g., a catalytic domain, a ligand
binding domain, or a protein-protein interaction domain), or a
polypeptide fragment of the selected protein of interest.
[0077] Dronpa nucleic acid and protein sequences may be derived
from corals of the genus Pectiniidae. A number of Dronpa nucleic
acid and protein sequences are known. Representative Dronpa
sequences are presented in SEQ ID NOS:1-4 and additional
representative sequences are listed in the National Center for
Biotechnology Information (NCBI) database. See, for example, NCBI
entries: Accession Nos. AB180726, ADE48854, BAD72874.1, 2IOV_D,
2IOV_C, 2IOV_B, 2IOV_A, 2POX_D, 2POX_C, 2POX_B, 2POX_A, AED56657,
AED56658, AED56659, and AED56660; all of which sequences (as
entered by the date of filing of this application) are herein
incorporated by reference. Any of these sequences or a variant
thereof comprising a sequence having at least about 80-100%
sequence identity thereto, including any percent identity within
this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be
used to construct a fusion protein, as described herein.
[0078] Padron nucleic acid and protein sequences may be derived
from Echinophyllia sp. SC22. A number of Padron nucleic acid and
protein sequences are known. Representative Padron sequences are
presented in SEQ ID NO:5 and SEQ ID NO:6. Additional representative
sequences are listed in the National Center for Biotechnology
Information (NCBI) database. See, for example, NCBI entries:
Accession Nos. ACL36360, ACL98050, EU983551, FJ014613, 3ZUL_A,
3ZUL_B, 3ZUL_C, 3ZUL_D, 3ZUL_E, 3ZUL_F, 3ZUJ_A, 3ZUJ_B, 3ZUJ_C,
3ZUJ_D, 3ZUJ_E, 3ZUJ_F, 3ZUF_A, 3ZUF_B, 3ZUF_C, 3ZUF_D, 3ZUF_E, and
3ZUF_F; all of which sequences (as entered by the date of filing of
this application) are herein incorporated by reference. Any of
these sequences or a variant thereof comprising a sequence having
at least about 80-100% sequence identity thereto, including any
percent identity within this range, such as 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence
identity thereto, can be used to construct a fusion protein, as
described herein.
[0079] RsTagRFP nucleic acid and protein sequences may be derived
from Discosoma sp. A number of rsTagRFP nucleic acid and protein
sequences are known. Representative rsTagRFP sequences are
presented in SEQ ID NO:7 and SEQ ID NO:8. Additional representative
sequences are listed in the National Center for Biotechnology
Information (NCBI) database. See, for example, NCBI entries:
Accession Nos. 3U8C_A, 3U8C_B, 3U8C_C, 3U8C_D, 3U8A_A, 3U8A_B,
3U8A_C, 3U8A_D; all of which sequences (as entered by the date of
filing of this application) are herein incorporated by reference.
Any of these sequences or a variant thereof comprising a sequence
having at least about 80-100% sequence identity thereto, including
any percent identity within this range, such as 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence
identity thereto, can be used to construct a fusion protein, as
described herein.
[0080] MApple nucleic acid and protein sequences may be derived
from Discosoma sp. A number of mApple nucleic acid and protein
sequences are known. Representative mApple sequences are presented
in SEQ ID NO:9 and SEQ ID NO:10. Additional representative
sequences are listed in the National Center for Biotechnology
Information (NCBI) database. See, for example, NCBI entries:
Accession Nos. ABC66097, DQ336160; all of which sequences (as
entered by the date of filing of this application) are herein
incorporated by reference. Any of these sequences or a variant
thereof comprising a sequence having at least about 80-100%
sequence identity thereto, including any percent identity within
this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be
used to construct a fusion protein, as described herein.
[0081] The photochromic polypeptides and other polypeptides
included in the fusion construct may be connected directly to each
other by peptide bonds or may be separated by intervening amino
acid sequences. The fusion polypeptides may also contain sequences
exogenous to the photochromic polypeptides or the selected protein
of interest. For example, the fusion may include targeting or
localization sequences, tag sequences, sequences of other
fluorescent proteins (e.g., with fluorescence characteristics that
differ from other photochromic proteins in the fusion protein), or
other chromophores. Moreover, the fusion may contain sequences from
multiple photochromic proteins, or variants thereof, and/or
non-photochromic proteins. For example, the fusion protein may
comprise two or more photochromic polypeptides, which can be the
same or different (e.g., two or more Dronpa 145K or Dronpa 145N
polypeptides, or a Dronpa 145K polypeptide and a Dronpa 145N
polypeptide simultaneously in the same fusion). Alternatively, the
fusion protein may comprise only one photochromic polypeptide,
which can be a wild-type photochromic polypeptide, or a variant
thereof.
[0082] In certain embodiments, the fusion protein can be
represented by the formula NH.sub.2-A-D-L-X-B-COOH or
NH.sub.2-A-X-L-D-B-COOH, wherein: D is an amino acid sequence of a
photochromic protein or a variant or polypeptide fragment thereof;
L is an optional linker amino acid sequence; X is an amino acid
sequence of a selected polypeptide of interest; A is an optional
N-terminal amino acid sequence; and B is an optional C-terminal
amino acid sequence.
[0083] In other embodiments, the fusion protein can be represented
by the formula NH.sub.2-A-D.sub.1-L-X-L-D.sub.2-B-COOH, wherein:
D.sub.1 and D.sub.2 are amino acid sequences of a photochromic
protein or a variant or polypeptide fragment thereof; L is an
optional linker amino acid sequence; X is an amino acid sequence of
a selected polypeptide of interest; A is an optional N-terminal
amino acid sequence; and B is an optional C-terminal amino acid
sequence. In fusion proteins comprising two photochromic
polypeptides, the photochromic polypeptides D.sub.1 and D.sub.2 can
be the same or different. For example, the fusion protein may
comprise two Dronpa 145N polypeptides, or two Dronpa 145K
polypeptides, or a Dronpa 145K polypeptide and a Dronpa 145N
polypeptide. Where more than one linker is present in the fusion,
the linkers can also be the same or different.
[0084] Linker amino acid sequence(s) -L- will typically be short,
e.g., 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1). Examples include
short peptide sequences which facilitate cloning, poly-glycine
linkers (Gly.sub.n where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more),
histidine tags (His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more),
linkers composed of glycine and serine residues ([Gly-Ser].sub.n,
[Gly-Gly-Ser-Gly].sub.n (SEQ ID NO:11), [Gly-Gly-Gly-Gly-Ser].sub.n
(SEQ ID NO:12), and [Ser-Ala-Gly-Gly].sub.n (SEQ ID NO:13), wherein
n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more), GSAT,
SEG, and Z-EGFR linkers. Linkers may include restriction sites,
which aid cloning and manipulation. Other suitable linker amino
acid sequences will be apparent to those skilled in the art. (See
e.g., Argos (1990) J. Mol. Biol. 211(4):943-958; Crasto et al.
(2000) Protein Eng. 13:309-312; George et al. (2002) Protein Eng.
15:871-879; Arai et al. (2001) Protein Eng. 14:529-532; and the
Registry of Standard Biological Parts
(partsregistry.org/Protein_domains/Linker).
[0085] -A- is an optional N-terminal amino acid sequence. This will
typically be short, e.g., 40 or fewer amino acids (i.e., 40, 39,
38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,
21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, or 1). Examples include leader sequences to direct protein
localization, or short peptide sequences or tag sequences, which
facilitate cloning or purification (e.g., a histidine tag His.sub.n
where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal
amino acid sequences will be apparent to those skilled in the
art.
[0086] -B- is an optional C-terminal amino acid sequence. This will
typically be short, e.g., 40 or fewer amino acids (i.e., 40, 39,
38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,
21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, or 1). Examples include sequences to direct protein
localization, short peptide sequences or tag sequences, which
facilitate cloning or purification (e.g., His.sub.n where n=3, 4,
5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein
stability. Other suitable C-terminal amino acid sequences will be
apparent to those skilled in the art.
[0087] In certain embodiments, tag sequences are located at the
N-terminus or C-terminus of the fusion protein. Exemplary tags that
can be used in the practice of the invention include a His-tag, a
Strep-tag, a TAP-tag, an S-tag, an SBP-tag, an Arg-tag, a
calmodulin-binding peptide tag, a cellulose-binding domain tag, a
DsbA tag, a c-myc tag, a glutathione S-transferase tag, a FLAG tag,
a HAT-tag, a maltose-binding protein tag, a NusA tag, and a
thioredoxin tag.
[0088] In certain embodiments, the fusion protein comprises a
targeting sequence. Exemplary targeting sequences that can be used
in the practice of the invention include a secretory protein signal
sequence, a membrane protein signal sequence, a nuclear
localization sequence, a nucleolar localization signal sequence, an
endoplasmic reticulum localization sequence, a peroxisome
localization sequence, a mitochondrial localization sequence, and a
protein-protein interaction motif sequence. Examples of targeting
sequences include those targeting the nucleus (e.g., KKKRK, SEQ ID
NO:14), mitochondrion (e.g., MLRTSSLFTRRVQPSLFRNILRLQST, SEQ ID
NO:15), endoplasmic reticulum (e.g., KDEL, SEQ ID NO:16),
peroxisome (e.g., SKL), synapses (e.g., S/TDV or fusion to GAP 43,
kinesin or tau), plasma membrane (e.g., CaaX (SEQ ID NO:17) where
"a" is an aliphatic amino acid, CC, CXC, CCXX (SEQ ID NO:18) at
C-terminus), or protein-protein interaction motifs (e.g., SH2, SH3,
PDZ, WW, RGD, Src homology domain, DNA-binding domain, SLiMs).
[0089] In another aspect, the invention includes a method for
controlling the activity of a selected polypeptide of interest with
light. The method comprises (i) preparing a fusion protein
comprising a first photochromic polypeptide connected to the
N-terminus of the selected polypeptide of interest and a second
photochromic polypeptide connected to the C-terminus of the
selected polypeptide of interest; (ii) illuminating the fusion
protein with light at a wavelength that induces intramolecular
dimerization of the first photochromic polypeptide and the second
photochromic polypeptide (e.g., about 405 nm for some fusions with
Dronpa 145N or 145K), such that the activity of the selected
polypeptide of interest is inactivated. In certain embodiments, the
method further comprises illuminating the fusion protein with light
that induces dissociation of the first photochromic polypeptide
from the second photochromic polypeptide (e.g., about 480-500 nm
for some fusions with Dronpa 145N or 145K), such that the activity
of the selected polypeptide is restored. Localization of the
selected polypeptide as well as inactivation of the selected
polypeptide can be visualized by detecting fluorescence of the
fusion protein resulting from intramolecular dimerization of the
first photochromic polypeptide and the second photochromic
polypeptide in the fusion protein. Inactivation of the selected
polypeptide can further be assessed by measuring the activity of
the selected polypeptide.
[0090] In another aspect, the invention includes a method for
controlling the localization of a selected polypeptide of interest
with light. The method comprises (i) preparing a first fusion
protein comprising a photochromic polypeptide connected to a
targeting sequence; (ii) preparing a second fusion protein
comprising a photochromic polypeptide connected to the selected
polypeptide of interest; (iii) introducing the first photochromic
fusion and the second fusion protein into a cell, wherein the
localization sequence targets the first fusion protein to a
particular subcellular location; (iv) and illuminating the fusion
proteins with light at a wavelength that induces oligomerization of
the photochromic polypeptide in the first fusion protein with the
photochromic polypeptide in the second fusion protein (e.g., about
405 nm for some fusions with Dronpa 145N or 145K), such that the
selected polypeptide of interest accumulates at the subcellular
location. In certain embodiments, the method further comprises
illuminating the fusion proteins with light that induces
dissociation of the photochromic polypeptides (e.g., about 480-500
nm for some fusions with Dronpa 145N or 145K), such that the
selected polypeptide of interest in the second fusion protein is
released from the subcellular location. Localization of the
selected polypeptide of interest can be visualized by detecting
fluorescence of the fusion proteins resulting from the
oligomerization of the photochromic polypeptides.
[0091] In another aspect, the invention includes a method for
controlling the localization of a selected polypeptide of interest
with light. The method comprises: (i) preparing a fusion protein
comprising a photochromic polypeptide, a targeting sequence, and
the selected polypeptide of interest; (ii) introducing the fusion
protein into a cell, wherein the localization sequence targets the
fusion protein to a particular subcellular location; and (iii)
illuminating the fusion protein with light at a wavelength that
induces oligomerization of the photochromic polypeptide in the
fusion protein with photochromic polypeptides in other fusion
proteins (e.g., about 405 nm for some fusions with Dronpa 145N or
145K), said other fusion proteins comprising the selected
polypeptide of interest, such that the selected polypeptide of
interest accumulates at the subcellular location. In certain
embodiments, the method further comprises illuminating the fusion
protein with light at a wavelength that induces dissociation of the
photochromic polypeptides (e.g., about 480-500 nm for some fusions
with Dronpa 145N or 145K), such that the selected polypeptide of
interest in the fusion protein is released from the subcellular
location. Localization of the selected polypeptide of interest can
be visualized by detecting fluorescence of the fusion protein
resulting from the oligomerization with photochromic polypeptides
of the other fusion proteins.
[0092] In the practice of the invention, the fluorescence of fusion
proteins can be monitored by any suitable method. For example,
fluorescence of fusion proteins can be detected by a fluorimeter, a
fluorescence microscope, a fluorescence microplate reader, a
fluorometric imaging plate reader, or fluorescence-activated cell
sorting.
[0093] B. Production of Fusion Proteins
[0094] Fusion proteins can be produced in any number of ways, all
of which are well known in the art. In one embodiment, the fusion
proteins are generated using recombinant techniques. One of skill
in the art can readily determine nucleotide sequences that encode
the desired polypeptides using standard methodology and the
teachings herein. Oligonucleotide probes can be devised based on
the known sequences and used to probe genomic or cDNA libraries.
The sequences can then be further isolated using standard
techniques and, e.g., restriction enzymes employed to truncate the
gene at desired portions of the full-length sequence. Similarly,
sequences of interest can be isolated directly from cells and
tissues containing the same, using known techniques, such as phenol
extraction and the sequence further manipulated to produce the
desired truncations. See, e.g., Sambrook et al., supra, for a
description of techniques used to obtain and isolate DNA.
[0095] The sequences encoding polypeptides can also be produced
synthetically, for example, based on the known sequences. The
nucleotide sequence can be designed with the appropriate codons for
the particular amino acid sequence desired. The complete sequence
is generally assembled from overlapping oligonucleotides prepared
by standard methods and assembled into a complete coding sequence.
See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)
Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311;
Stemmer et al. (1995) Gene 164:49-53.
[0096] Recombinant techniques are readily used to clone sequences
encoding polypeptides useful in the claimed fusion proteins that
can then be mutagenized in vitro by the replacement of the
appropriate base pair(s) to result in the codon for the desired
amino acid. Such a change can include as little as one base pair,
effecting a change in a single amino acid, or can encompass several
base pair changes. Alternatively, the mutations can be effected
using a mismatched primer that hybridizes to the parent nucleotide
sequence (generally cDNA corresponding to the RNA sequence), at a
temperature below the melting temperature of the mismatched duplex.
The primer can be made specific by keeping primer length and base
composition within relatively narrow limits and by keeping the
mutant base centrally located. See, e.g., Innis et al, (1990) PCR
Applications: Protocols for Functional Genomics; Zoller and Smith,
Methods Enzymol. (1983) 100:468. Primer extension is effected using
DNA polymerase, the product cloned and clones containing the
mutated DNA, derived by segregation of the primer extended strand,
selected. Selection can be accomplished using the mutant primer as
a hybridization probe. The technique is also applicable for
generating multiple point mutations. See, e.g., Dalbie-McFarland et
al. Proc. Natl. Acad. Sci USA (1982) 79:6409.
[0097] Once coding sequences have been isolated and/or synthesized,
they can be cloned into any suitable vector or replicon for
expression. (See, also, Examples). As will be apparent from the
teachings herein, a wide variety of vectors encoding modified
polypeptides can be generated by creating expression constructs
which operably link, in various combinations, polynucleotides
encoding polypeptides having deletions or mutations therein.
[0098] Numerous cloning vectors are known to those of skill in the
art, and the selection of an appropriate cloning vector is a matter
of choice. Examples of recombinant DNA vectors for cloning and host
cells which they can transform include the bacteriophage .lamda.
(E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative
bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells).
See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et
al., supra; B. Perbal, supra.
[0099] Insect cell expression systems, such as baculovirus systems,
can also be used and are known to those of skill in the art and
described in, e.g., Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987). Materials and methods
for baculovirus/insect cell expression systems are commercially
available in kit form from, inter alia, Invitrogen, San Diego
Calif. ("MaxBac" kit).
[0100] Plant expression systems can also be used to produce the
fusion proteins described herein. Generally, such systems use
virus-based vectors to transfect plant cells with heterologous
genes. For a description of such systems see, e.g., Porta et al.,
Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol.
(1994) 139:1-22.
[0101] Viral systems, such as a vaccinia based
infection/transfection system, as described in Tomei et al., J.
Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)
74:1103-1113, will also find use with the present invention. In
this system, cells are first transfected in vitro with a vaccinia
virus recombinant that encodes the bacteriophage T7 RNA polymerase.
This polymerase displays exquisite specificity in that it only
transcribes templates bearing T7 promoters. Following infection,
cells are transfected with the DNA of interest, driven by a T7
promoter. The polymerase expressed in the cytoplasm from the
vaccinia virus recombinant transcribes the transfected DNA into RNA
that is then translated into protein by the host translational
machinery. The method provides for high level, transient,
cytoplasmic production of large quantities of RNA and its
translation product(s).
[0102] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator (collectively referred to herein as "control"
elements), so that the DNA sequence encoding the desired
polypeptide is transcribed into RNA in the host cell transformed by
a vector containing this expression construction. The coding
sequence may or may not contain a signal peptide or leader
sequence. With the present invention, both the naturally occurring
signal peptides and heterologous sequences can be used. Leader
sequences can be removed by the host in post-translational
processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437;
4,338,397. Such sequences include, but are not limited to, the TPA
leader, as well as the honey bee mellitin signal sequence.
[0103] Other regulatory sequences may also be desirable which allow
for regulation of expression of the protein sequences relative to
the growth of the host cell. Such regulatory sequences are known to
those of skill in the art, and examples include those which cause
the expression of a gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Other types of regulatory elements may also be
present in the vector, for example, enhancer sequences.
[0104] The control sequences and other regulatory sequences may be
ligated to the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector that already contains the control sequences and
an appropriate restriction site.
[0105] In some cases it may be necessary to modify the coding
sequence so that it may be attached to the control sequences with
the appropriate orientation; i.e., to maintain the proper reading
frame. Mutants or analogs may be prepared by the deletion of a
portion of the sequence encoding the protein, by insertion of a
sequence, and/or by substitution of one or more nucleotides within
the sequence. Techniques for modifying nucleotide sequences, such
as site-directed mutagenesis, are well known to those skilled in
the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I
and II, supra; Nucleic Acid Hybridization, supra.
[0106] The expression vector is then used to transform an
appropriate host cell. A number of mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others.
Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and
Streptococcus spp., will find use with the present expression
constructs. Yeast hosts useful in the present invention include
inter alia, Saccharomyces cerevisiae, Candida albicans, Candida
maltosa, Hansenula polymorphs, Kluyveromyces fragilis,
Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,
Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for
use with baculovirus expression vectors include, inter alia, Aedes
aegypti, Autographa califormica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
[0107] Depending on the expression system and host selected, the
fusion proteins of the present invention are produced by growing
host cells transformed by an expression vector described above
under conditions whereby the protein of interest is expressed. The
selection of the appropriate growth conditions is within the skill
of the art.
[0108] In one embodiment, the transformed cells secrete the
polypeptide product into the surrounding media. Certain regulatory
sequences can be included in the vector to enhance secretion of the
protein product, for example using a tissue plasminogen activator
(TPA) leader sequence, an interferon (.gamma. or .alpha.) signal
sequence or other signal peptide sequences from known secretory
proteins. The secreted polypeptide product can then be isolated by
various techniques described herein, for example, using standard
purification techniques such as but not limited to, hydroxyapatite
resins, column chromatography, ion-exchange chromatography,
size-exclusion chromatography, electrophoresis, HPLC,
immunoadsorbent techniques, affinity chromatography,
immunoprecipitation, and the like.
[0109] Alternatively, the transformed cells are disrupted, using
chemical, physical or mechanical means, which lyse the cells yet
keep the recombinant polypeptides substantially intact.
Intracellular proteins can also be obtained by removing components
from the cell wall or membrane, e.g., by the use of detergents or
organic solvents, such that leakage of the polypeptides occurs.
Such methods are known to those of skill in the art and are
described in, e.g., Protein Purification Applications: A Practical
Approach, (Simon Roe, Ed., 2001).
[0110] For example, methods of disrupting cells for use with the
present invention include but are not limited to: sonication or
ultrasonication; agitation; liquid or solid extrusion; heat
treatment; freeze-thaw; desiccation; explosive decompression;
osmotic shock; treatment with lytic enzymes including proteases
such as trypsin, neuraminidase and lysozyme; alkali treatment; and
the use of detergents and solvents such as bile salts, sodium
dodecylsulphate, Triton, NP40 and CHAPS. The particular technique
used to disrupt the cells is largely a matter of choice and will
depend on the cell type in which the polypeptide is expressed,
culture conditions and any pre-treatment used.
[0111] Following disruption of the cells, cellular debris is
removed, generally by centrifugation, and the intracellularly
produced polypeptides are further purified, using standard
purification techniques such as but not limited to, column
chromatography, ion-exchange chromatography, size-exclusion
chromatography, electrophoresis, HPLC, immunoadsorbent techniques,
affinity chromatography, immunoprecipitation, and the like.
[0112] For example, one method for obtaining the intracellular
polypeptides of the present invention involves affinity
purification, such as by immunoaffinity chromatography using
antibodies (e.g., previously generated antibodies), or by lectin
affinity chromatography. Particularly preferred lectin resins are
those that recognize mannose moieties such as but not limited to
resins derived from Galanthus nivalis agglutinin (GNA), Lens
culinaris agglutinin (LCA or lentil lectin), Pisum sativum
agglutinin (PSA or pea lectin), Narcissus pseudonarcissus
agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of
a suitable affinity resin is within the skill in the art. After
affinity purification, the polypeptides can be further purified
using conventional techniques well known in the art, such as by any
of the techniques described above.
[0113] Polypeptides can be conveniently synthesized chemically, for
example by any of several techniques that are known to those
skilled in the peptide art. In general, these methods employ the
sequential addition of one or more amino acids to a growing peptide
chain. Normally, either the amino or carboxyl group of the first
amino acid is protected by a suitable protecting group. The
protected or derivatized amino acid can then be either attached to
an inert solid support or utilized in solution by adding the next
amino acid in the sequence having the complementary (amino or
carboxyl) group suitably protected, under conditions that allow for
the formation of an amide linkage. The protecting group is then
removed from the newly added amino acid residue and the next amino
acid (suitably protected) is then added, and so forth. After the
desired amino acids have been linked in the proper sequence, any
remaining protecting groups (and any solid support, if solid phase
synthesis techniques are used) are removed sequentially or
concurrently, to render the final polypeptide. By simple
modification of this general procedure, it is possible to add more
than one amino acid at a time to a growing chain, for example, by
coupling (under conditions which do not racemize chiral centers) a
protected tripeptide with a properly protected dipeptide to form,
after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co.,
Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.
Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254,
for solid phase peptide synthesis techniques; and M. Bodansky,
Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984) and
E. Gross and J. Meienhofer, Eds., The Peptides: Analysis,
Synthesis, Biology, Vol. 1, for classical solution synthesis. These
methods are typically used for relatively small polypeptides, i.e.,
up to about 50-100 amino acids in length, but are also applicable
to larger polypeptides.
[0114] Typical protecting groups include t-butyloxycarbonyl (Boc),
9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);
p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);
biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,
isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl,
isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
[0115] Typical solid supports are cross-linked polymeric supports.
These can include divinylbenzene cross-linked-styrene-based
polymers, for example, divinylbenzene-hydroxymethylstyrene
copolymers, divinylbenzene-chloromethylstyrene copolymers and
divinylbenzene-benzhydrylaminopolystyrene copolymers.
[0116] Polypeptide analogs can also be chemically prepared by other
methods such as by the method of simultaneous multiple peptide
synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985)
82:5131-5135; U.S. Pat. No. 4,631,211.
[0117] C. Kits
[0118] Fusion proteins or nucleic acids encoding them can be
provided in kits with suitable instructions and other necessary
reagents for preparing or using the fusion proteins, as described
above. The kit may contain in separate containers fusion proteins,
or recombinant constructs for producing fusion proteins, and/or
cells (either already transfected or separate). Additionally,
instructions (e.g., written, tape, VCR, CD-ROM, DVD, etc.) for
using the fusion proteins may be included in the kit. The kit may
also contain other packaged reagents and materials (e.g.,
transfection reagents, buffers, media, and the like).
[0119] D. Applications
[0120] The fusion proteins of the invention provide useful tools
for spatially and temporally controlling protein activity with
light and will find numerous applications in basic research and
development. In particular, fusion proteins can be designed to
block or induce activities of proteins of interest, control their
interactions with other macromolecules, or direct their subcellular
localization. Because fusion proteins can potentially be used to
control diverse cellular processes with light, they will be
especially useful in the study of protein function in physiological
processes and disease mechanisms.
III. EXPERIMENTAL
[0121] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0122] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Example 1
Optical Control of Protein Activity by Fusion to Fluorescent
Protein Domains
Introduction
[0123] Here, we describe the discovery of an engineered protein
interaction that is controlled by cyan light and requires no
cofactors. We use this light-controlled association to develop a
simple generalizable design for light-inducible proteins. We
created a fluorescent light-inducible protein design in which
Dronpa domains are fused to both termini of an enzyme domain. In
the dark, the Dronpa domains associate and cage the protein, but
light induces Dronpa dissociation and activates the protein. This
method enabled optical control over guanine nucleotide exchange
factor (GEF) and protease domains without extensive screening. Our
findings extend the applications of fluorescent proteins from
exclusively sensing functions to also encompass optogenetic
control.
[0124] Dronpa is a monomeric fluorescent protein (FP) derived from
the tetrameric parent protein, 22G, isolated from a Pectiniidae
genus coral (Ando et al. (2004) Science 306:1370-1373).
Fluorescence of Dronpa switches off under cyan light (.about.500
nm) and switches on under violet light (.about.400 nm) (Ando et
al., supra). With off-photoswitching, .beta. strand 7 near the
chromophore becomes flexible (Mizuno et al. (2008) Proc. Natl.
Acad. Sci. U.S.A. 105:9227-9232); this strand forms part of the
cross-dimer interface in the tetrameric parent (Mizuno et al.
(2008), supra). A Dronpa mutant with Lys.sup.145 on .beta. strand 7
changed to Asn (Dronpa145N) is tetrameric at low micromolar
concentrations, but dilution promotes monomerization and
facilitates off-photoswitching (Mizuno et al. (2010) Photochem.
Photobiol. Sci. 9: 239-248). This suggests that multimerization
inhibits conformation changes associated with off-photoswitching.
We hypothesized, conversely, that conformation changes occurring
during off-photoswitching might promote monomerization, whereas
on-photoswitching might promote multimerization (FIG. 1A).
[0125] Materials and Methods
[0126] DNA Construction
[0127] pcDNA3-mNeptune1-fascin was a gift of Michael W. Davidson
(Florida State University, Tallahassee). tdTomato and mCherry
plasmids were gifts of Nathan Shaner and Roger Y. Tsien (UCSD).
Dronpa145K and Dronpa145N were synthesized by polymerase chain
reaction (PCR) of overlapping oligonucleotides and cloned into
pNCS, a constitutive bacterial expression vector with a
six-consecutive-histidine tag at its N-terminus for purification
and BamHI and EcoRI sites for insert cloning (Muller et al. (2008)
ChemBioChem 9:2029-2038; herein incorporated by reference). A
construct encoding amino acids 1234-1428 of the human intersectin
DH domain (Entrez Gene ID 6453) was synthesized by PCR from
overlapping oligonucleotides and cloned into the mammalian
expression vector pcDNA3 (Invitrogen). Plasmids containing HCV
protease and substrate sequences and mRuby2 were previously
described (Faix et al. (2009) Int. J. Biochem. Cell Biol.
41:1656-1664; Lam et al. (2012) Nat. Methods 9:1005-1012; herein
incorporated by reference).
[0128] In addition to pNCS-Dronpa145K and pNCS-Dronpa145N described
above, to create other bacterial expression constructs for native
polyacrylamide gel electrophoresis, mRuby2, tdTomato, and DsRed2
open reading frames (ORFs) were amplified from pBAD-tdTomato,
pcDNA3-mRuby2, and pDsRed2-N1 (Clontech), respectively, and cloned
into pNCS. pNCS-tdDronpa145K-Dronpa145N was created by
recombination of a PCR-amplified Dronpa145K ORF with BamHI-digested
pNCS-Dronpa145N using the In-Fusion recombinase (Clontech).
[0129] To create mammalian expression plasmids for fusions of
Dronpa and intersectin domains, PCR fragments encoding Dronpa and
intersectin DH domains and the Kras4B CAAX sequence
(KMSKDGKKKKKKSKTKCVIM, SEQ ID NO:21) were amplified from the above
plasmids or from overlapping oligos (for the CAAX sequence), then
assembled in a second PCR reaction and cloned into pcDNA3. To
create plasmids coexpressing mCherry-substrate-CAAX and fusions of
Dronpa and HCV NS4A/NS3 protease, the lentiviral vector pLL3.7
(Addgene) was first modified to reduce its size by replacing the
untranslated sequence between PvuII and BspEI sites upstream of the
3' long terminal repeat with a more compact sequence containing
only the polypyrimidine tract and integrase att site necessary for
reverse transcription and integration, creating pLL3.7m. Then a
fusion of mCherry, the NS4A/NS4B substrate sequence, and CAAX was
assembled by overlapping PCR and inserted between NheI and EcoRV
sites downstream of the CMV promoter by ligation, creating
pLL3.7m-mCherry-substrate-CAAX. Finally, PCR fragments encoding a
minimal CMV promoter, Dronpa and HCV NS4A/NS3 protease domains, and
SV40 polyadenylation signals from pcDNA3 were assembled and
inserted between the NotI and XbaI sites of
pLL3.7m-mCherry-substrate-CAAX by the In-Fusion recombinase. This
created an expression cassette adjacent to and in the opposite
transcriptional direction from the original CMV promoter.
[0130] In Vitro Protein Characterization and Photoswitching
[0131] Bacterial expression plasmids for Dronpa145K, Dronpa145N,
tdDronpa145K-Dronpa145N, mRuby, tdTomato, and dsRed2 were
transformed into chemically competent Escherichia coli strain
DH5.alpha. for expression. A single colony was inoculated into 100
ml of Luria-Bertani (LB) broth containing 50 .mu.g ml.sup.-1
ampicillin and incubated overnight at 37.degree. C. The cultures
were further incubated at room temperature for another 24 hours,
then fluorescent protein purification from bacterial lysates was
performed by polyhistidine affinity purification as previously
described (Muller et al., supra). Protein concentrations were
estimated by absorbance spectrophotometry and purity was verified
by SDS-PAGE. For characterization of baseline oligomerization
state, 5 .mu.L each of 100 .mu.M, 20 .mu.M, or 10 .mu.M of
Dronpa145K or Dronpa145N were run on a 4-16% Bis-Tris native PAGE
gel (Invitrogen) with dark cathode buffer alongside 5 .mu.L each of
20 .mu.M mRuby2, tdTomato, and dsRed2 as size controls.
[0132] For in vitro photoswitching, purified Dronpa145N and tandem
dimer Dronpa145K-Dronpa145N proteins were diluted to 100 .mu.M.
Proteins in a 0.2-mL PCR tube were switched off by placement
between two cyan LEDs mounted 1 inch apart for 30 minutes (505/30
nm, 170 mW, Thorlab). The fluorescence recovery was conducted by
illumination with two similarly mounted UV LEDs for 30 seconds
(405/20 nm, 470 mW, Thorlab), followed by incubation at room
temperature for 30 minutes. The switching efficiency was estimated
by measuring the fluorescence of 1-.mu.L protein aliquots using a
Safire 2 monochromator-based fluorescence spectrophotometer
(TECAN). In parallel, 2.5 .mu.L (Dronpa145N) or 5 .mu.L
(tdDronpa145K-Dronpa145N) of the protein in each condition were
loaded on a 4-16% Bis-Tris native PAGE gel (Invitrogen) with dark
cathode buffer. 5 .mu.L each of 20 .mu.M mRuby, tdTomato, and
dsRed2 were loaded as size controls.
[0133] Cell Culture and Transfection
[0134] Cells were maintained in high glucose Dulbecco's Modified
Eagle Medium (DMEM, HyClone) supplemented with 10% fetal bovine
serum (FBS, Invitrogen) and 2 mM glutamine (Sigma) at 37.degree. C.
in air with 5% carbon dioxide. Hela cells were transfected at
75-90% confluency with Lipofectamine 2000 (Invitrogen) in 33-mm
coverglass-bottom dishes (In Vitro Scientific). Transfections were
carried out according to manufacturer's instructions, except that
amounts of DNA and transfection reagent were halved to reduce cell
toxicity. NIH 3T3 cells (5-7.times.10.sup.4) were plated directly
in a transfection solution containing DNA plasmids and
Lipofectamine 2000 in 33-mm coverglass-bottom dishes. Amounts of
DNA and transfection reagent were reduced to 1/5 of the
manufacture-recommended amount for a 33 mm culture. For both HeLa
and NIH 3T3 cells, the medium was refreshed 4-6 hours after
transfection. HEK293 cells were grown in 8 well-chambered
coverglass (Nunc) and transfected at 75-90% confluency using
Lipofectamine LTX (Invitrogen) according to the manufacturer's
instructions.
[0135] Membrane Translocation and Protein Uncaging
[0136] In the translocation assay, Hela cells were imaged in PBS at
room temperature 12-36 hours after transfection. Imaging was
performed with a C-Apochromat 40.times.1.2 numerical aperture (NA)
water-immersion objective on a Zeiss Axiovert 200 M with a Ludl
excitation filter controlled by a Ludl MAC 5000 controller, using a
Hamamatsu Orca ER Firewire camera. All instruments were controlled
by a 2.5 Ghz MacBook Pro computer running Micro-manager 1.4
software in Mac OS 10.6.8. Illumination was provided by a 120-W
metal-halide light source (Exfo) passed through a 1-m liquid light
guide with a 3 mm core. Dronpa was imaged with a 10% neutral
density filter, a 485/30-nm excitation filter, a 505-nm dichroic
mirror, and a 525/40 nm emission filter. Neptune was imaged with a
10% neutral density filter, a 560/40 nm excitation filter, a 585 nm
dichroic mirror, and a 630/75 emission filter. Dronpa was
photoswitched off by illumination using the Dronpa channel
excitation and dichroic filters and no neutral density filter for
the indicated times. The light intensity was measured to be 4.7 W
cm.sup.-2. Images were acquired within 2 minutes after
photoswitching to report Dronpa photoswitching and mNeptune
movements. Light passed through a 10% neutral density filter and a
405/20 nm filter and a 440 nm dichroic mirror was used to recover
Dronpa fluorescence. Images were acquired immediately to report
Dronpa recovery and 5 minutes later to report the mNeptune
movements.
[0137] For intersectin experiments, NIH 3T3 cells were incubated in
serum-free DMEM media for 5-9 hours beginning 32-48 hours after
transfection. Cells were then imaged in HBSS at room temperature as
described above. Dronpa was photoswitched as described above. Cells
were imaged at intervals 5-10 minutes apart for up to 1 hour.
[0138] For protease experiments, HEK293 cells were imaged 16 hours
after transfection in HBSS with 2% B27 (Invitrogen), 1 mM sodium
pyruvate, and 10 mM HEPES pH 7.2 in a TC CU109 chamber (Chamide)
heated to 37.degree. C. Imaging was done with an Olympus 40.times.
1.15 NA water immersion objective on Olympus IX80 with a Ludl
excitation filter controlled by a Ludl MAC 5000 controller, using a
Hamamatsu Orca ER Firewire camera. All instruments were controlled
by a 3 GHz Dell Optiplex 755SFF computer running Micro-manager 1.4
software in Windows 7. Illumination was provided by a 120-W
metal-halide light source (Exfo) passed through a 1-m liquid light
guide with a 3 mm core. Dronpa was imaged with a 485/22 nm
excitation filter, 510 nm dichroic mirror, and 540/40 nm emission
filter. The mCherry was imaged with a 545/30 nm excitation filter,
570 nm dichroic mirror, and 605/50 nm emission filter. Dronpa was
photoswitched off by 10 seconds of illumination using the Dronpa
channel excitation and dichroic filters and no neutral density
filter. Images were acquired at 10 minutes, 30 minutes and 60
minutes after photoswitching.
[0139] Statistical Analysis
[0140] To determine statistical significance of light-dependent
filopodia induction, the Pearson chi-squared test was performed on
the distributions of the two observation outcomes of filopodia
induction or no filopodia induction between two treatment
conditions of illumination or no illumination. The null hypothesis
was that filopodia induction is independent of treatment condition.
A responding cell was defined as a cell with at least one new
filopodium per polygonal side.
[0141] Results
[0142] To determine if light could control Dronpa145N
multimerization, we performed native polyacrylamide gel
electrophoresis (PAGE). Dronpa145N was tetrameric at concentrations
from 10 to 100 .mu.M in the initial bright state, whereas wild-type
Dronpa (Dronpa145K for clarity; K, Lys) was monomeric (FIG. 5A).
Cyan illumination of 100 .mu.M Dronpa145N induced a shift from a
cyan-absorbing to a violet-absorbing species (FIG. 1C) and a loss
of green fluorescence (FIG. 5B), as previously described (Ando et
al. (2004) Science 306: 1370-1373). Simultaneously, Dronpa145N
redistributed from tetrameric toward monomeric species (FIG. 1B,
lane 2), implying that off-photoswitched Dronpa145N has a
dissociation constant exceeding 100 .mu.M. Violet light restored
cyan absorbance (FIG. 1C) and green fluorescence (FIG. 5B) and also
induced retetramerization (FIG. 1B, lane 3), indicating that
monomerization was not due to irreversible protein damage. These
results show that Dronpa145N interactions can be controlled by
light.
[0143] A dimer-to-monomer conversion might be more easily harnessed
to control protein activity than a tetramer-to-monomer conversion.
Given the lack of multimerization of Dronpa 145K, we explored
whether oligomerization of Dronpa145K and Dronpa145N could be
limited to dimerization. To achieve high effective concentrations
of Dronpa145K and Dronpa145N without driving Dronpa145N
tetramerization, we fused Dronpa145K in tandem to Dronpa145N via a
linker (K-N tandem dimer) (FIG. 1D). The effective concentration of
one domain relative to another on the same polypeptide has been
estimated at .about.70 .mu.M (Muller et al., supra). The K-N
construct migrated in native PAGE primarily as expected for a
tandem dimer (FIG. 1E). If the Dronpa domains were engaged in a
light-sensitive intramolecular interaction, illumination should
induce dissociation, resulting in a more elongated faster-migrating
conformation. Indeed, the tandem dimer migrated faster after cyan
illumination, and this process was reversed after violet
light-induced recovery (FIG. 1E). Expected transitions between
cyan- and violet-absorbing forms were again observed (FIG. 1F and
FIG. 5C). Thus, the K-N tandem dimer undergoes reversible
light-induced conformational changes consistent with dissociation
and reassociation of Dronpa domains.
[0144] To determine whether light-induced Dronpa145N dissociation
could occur in mammalian cells, we created two fusions: N-CAAX, a
fusion of Dronpa145N to the membrane-anchoring K-Ras C-terminal
farnesylation motif (CAAX box), and mNeptune-N, a fusion of the
far-red FP mNeptune to Dronpa145N (FIG. 2A) (Lin et al. (2009)
Chem. Biol. 16:1169-1179). Upon 10-fold relative overexpression of
N-CAAX to insure an excess of membrane-localized Dronpa, some
mNeptune-N was membrane-bound through Dronpa145N oligomerization
(FIGS. 2C and 2D). Cyan light switched off Dronpa fluorescence
(FIG. 2B) and resulted in the release of mNeptune from the membrane
(FIGS. 2C and 2D). Release required prolonged exposures (2 minutes,
metal halide lamp at 100% neutral density through a
40.times.1.2-numerical aperture lens) and was only partial, but
nevertheless indicated that light could induce Dronpa domain
dissociation in cells.
[0145] To find conditions for Dronpa domain dissociation that
require less light, we explored Dronpa145K-Dronpa145N
heterodimerization (FIG. 2E). Dronpa145K-CAAX (K-CAAX) was able to
recruit mNeptune-N to the membrane (FIG. 2G). Off-photoswitching of
membrane fluorescence was faster than with N-CAAX (FIG. 2F), and
release of mNeptune required only 20 seconds of illumination (FIGS.
2G and 2H). On-photoswitching of Dronpa by violet light induced
membrane re-localization of mNeptune-N (FIGS. 2G and 2H). Reversing
the positions of Dronpa domains by expressing N-CAAX and
mNeptune-Dronpa145K (mNeptune-K) did not result in a membrane
mNeptune signal (FIG. 6A), perhaps because tetramerization between
concentrated N-CAAX molecules outcompeted weaker heterodimerization
with mNeptune-K. Use of only monomeric Dronpa domains (K-CAAX and
mNeptune-K) also resulted in no membrane mNeptune (FIG. 6B), as
expected.
[0146] We hypothesized that we could use Dronpa to build
light-controllable single chain proteins. Specifically, we
hypothesized that protein functions could be blocked by fusing
Dronpa domains to the amino terminus (NT) and the carboxyl terminus
(CT) (FIG. 7A). Binding of the two Dronpa domains would "cage" the
protein in an inactive state by masking surfaces required for
binding interaction partners or substrates, similarly to
auto-inhibition of many kinases (Leonard et al. (2007) Cell
129:1037-1038), transcription factors (Pufall et al. (2002) Annu
Rev. Cell Dev. Biol. 18:421-462), and guanine nucleotide exchange
factors (GEFs) for monomeric guanosine triphosphatases (GTPases)
(Yu et al. (2010) Cell 140:246-256). Protein function could then be
induced by light-mediated dissociation of the Dronpa domains (FIG.
7A).
[0147] We first controlled the Cdc42 GEF intersectin, which can be
inactivated by terminal circularization (Yeh et al. (2007) Nature
447:596-600). We fused Dronpa145K or Dronpa145N at the NT of the
intersectin Dbl homology (DH) domain and Dronpa145N at the CT
followed by the CAAX sequence, creating K-I-N-CAAX and N-I-N-CAAX
(FIG. 7B). As catalytically active controls, we fused Dronpa145K to
either side of intersectin (K-I-CAAX and I-K-CAAX) (FIG. 7B). We
coexpressed these constructs in fibroblasts with a mNeptune-fascin
reporter to mark filopodia and lamellipodia (Adams (2004) Curr.
Opin. Cell Biol. 16:590-596). I-K-CAAX or K-I-CAAX robustly induced
filopodia and lamellipodia (FIGS. 7C and 7D), as expected for Cdc42
activation, which induces filopodia directly and lamellipodia
directly via the formin-family protein FMNL2 and indirectly via Rac
(Block et al. (2012) Curr. Biol. 22:1005-1012; Nishimura et al.
(2005) Nat. Cell Biol. 7:270-277). Cells expressing N-I-N-CAAX and
K-I-N-CAAX produced filopodia or lamellipodia at much lower
frequencies than I-K-CAAX or K-I-CAAX (FIGS. 7C and 7D). These
experiments were performed by transient transfection, which results
in variable expression levels. When designated as low, medium, or
high expressers by Dronpa fluorescence (FIGS. 8A and 8C), low
expressers, which included the majority of cells, exhibited basal
filopodia or lamellipodia infrequently (0% for N-I-N-CAAX and 8%
for K-I-N-CAAX) (FIGS. 8B and 8D). Thus, fusion of flanking Dronpa
domains cages intersectin activity effectively as long as higher
expression levels are avoided, similarly to tophototropin-based
photoactivable Rac (PA-Rac; Wu et al. (2011) Methods Enzymol.
497:393-407).
[0148] We next asked whether caged intersectins could mediate
filopodia or lamellipodia induction by light (FIGS. 3A and 3E).
Illumination with 490/20-nm light for 30 seconds switched off more
than 50% of the fluorescence in both N-I-N-CAAX- and
K-I-N-CAAX-transfected fibroblasts (FIGS. 3B and 3F). This light
dose induced abundant filopodia formation within 30 minutes in 78%
of cells expressing N-I-N-CAAX (FIG. 3C and FIG. 9D). This response
was light-dependent, as only 10% of cells expressing N-I-N-CAAX
formed filopodia in the same time interval without illumination
(P<0.0001 by Pearson .chi..sup.2 test) (FIGS. 9A and 9D). Cells
continued to exhibit filopodial mobility throughout 1 hour of
observation and did not show blebbing that might indicate
phototoxicity. Similarly, 90% of cells expressing K-I-N-CAAX formed
abundant filopodia within 30 minutes after illumination (FIG. 3G,
FIG. 9D), compared with 25% not exposed to light (P<0.0001 by
Pearson x.sup.2 test) (FIGS. 9B and 9D). Illumination of
K-CAAX-expressing cells did not induce filopodia (FIGS. 9C and 9D),
confirming that the effect is not due to light alone. These results
demonstrate that a protein caged by Dronpa fusion can be uncaged by
light.
[0149] We investigated whether caged intersectin constructs could
control filopodia formation with spatial or temporal specificity.
First, we performed local illumination (490/20-nm light for 30
seconds) to portions of cells expressing N-I-N-CAAX or K-IN-CAAX
and observed that filopodia appeared specifically in the
illuminated regions (FIGS. 3D and 3H). We next tested whether light
could induce filopodia in different locations at different times in
one cell. We applied a 30-second uncaging pulse of cyan light at
one subcellular region, a 3-second global recaging pulse of violet
light, and finally another 30-second uncaging pulse at a different
subcellular region. After the first uncaging pulse, filopodia
appeared in the first region, whereas after the global recaging and
second uncaging pulse, filopodia appeared in the second region
simultaneous with retraction in the first region (FIG. 10).
[0150] Whether Cdc42 activation can lengthen existing filopodia has
been unclear, as Cdc42 effectors that promote filopodia extension
rather than initiation have not been found. Rapid induction of
intersectin activity by light allowed us to address this question.
We observed that photouncaging of intersectin caused lengthening of
many preexisting filopodia (FIG. 11). This suggests that models in
which Cdc42 governs only filopodia initiation are incomplete (Faix
et al. (2009) Int. J. Biochem. Cell Biol. 41:1656-1664) and that
effectors may exist that promote filopodia extension analogous to
how FMNL2 promotes lamellipodia extension downstream of Cdc42
(Block et al. (2012) Curr. Biol. 22:1005-1112).
[0151] An attractive feature of our design is potential
generalizability. Other methods for optical control of single
polypeptides, such as fusion to xanthopsin or phototropin, require
extensive screening to achieve coupling of light-induced
conformational changes to protein activation and, thus, have been
applied to only a few targets (Fan et al. (2011) Biochemistry
50:1226-1237; Wu et al. (2009) Nature 461:104-108; Strickland et
al. (2010) Nat. Methods 7:623-626). Our caged protein design does
not require precise linkages; therefore, it should be more easily
generalizable. Proteases are a class of enzymes for which light
activation has not yet been achieved. Unlike GTPases or kinases,
proteases are not naturally regulated by membrane recruitment,
preventing the use of reversible membrane targeting methods to
control them. Hence, we investigated whether we could create a
light-inducible protease by fusion to Dronpa domains. We chose to
regulate the hepatitis C virus (HCV) NS3-4A protease because its
high sequence specificity and lack of overt toxicity allows
assessment of function in mammalian cells (Lin et al. (2008) Proc.
Natl. Acad. Sci. U.S.A. 105:7744-7749). Furthermore, it is composed
predominantly of .beta. strands and loops (Romano et al. (2010)
Proc. Natl. Acad. Sci. U.S.A. 107:20986-20991), providing a
structural contrast to the completely .alpha.-helical DH domain
(Snyder et al. (2002) Nat. Struct. Biol. 9:468-475).
[0152] We constructed a Dronpa145N-protease-Dronpa145N fusion
(N-protease-N) and, as a protease reporter, a fusion of mCherry,
the NS4A/NS4B cleavage site of HCV polypeptide, and the CAAX-box
farnesylation signal (mCherry-substrate-CAAX) (FIG. 4A). We
expected that mCherry fluorescence would be released from the
membrane into the cytosol by protease activity. Indeed, mCherry
signal was membrane-bound in cells expressing
mCherry-substrate-CAAX alone and cytoplasmic in cells coexpressing
a positive control Dronpa145K-protease (FIG. 4B). We then used
mCherry-substrate-CAAX to report light induction of N-protease-N.
After off-switching of Dronpa fluorescence, cells showed an
increase in cytosolic mCherry within 10 minutes, which continued to
increase over 60 minutes (FIG. 4C). This response required
illumination (FIG. 12A) and protease (FIG. 12B). Thus, the caged
protein design can be used to control an enzyme domain that is not
easily regulated by relocalization within the cell.
[0153] Since their discovery, FPs have seen widespread use
exclusively as sensing tools. We discovered that photochromic FPs
can have dual identities as optical sensors and light-controlled
actuators. We have translated this discovery into a simple design
for optically controllable proteins, which we propose to call
FLIPs, for fluorescent light-inducible proteins. FLIPs also serve
as their own reporters, as the photochromic FP domains report both
protein localization and activity state. Thus, our results place
photochromic FPs in a distinct central location in the optogenetic
toolbox, integrating both sensing and controlling functions in a
single protein class.
[0154] While the preferred embodiments of the invention have been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
Sequence CWU 1
1
211224PRTArtificialDronpa-145K 1Met Ser Val Ile Lys Pro Asp Met Lys
Ile Lys Leu Arg Met Glu Gly 1 5 10 15 Ala Val Asn Gly His Pro Phe
Ala Ile Glu Gly Val Gly Leu Gly Lys 20 25 30 Pro Phe Glu Gly Lys
Gln Ser Met Asp Leu Lys Val Lys Glu Gly Gly 35 40 45 Pro Leu Pro
Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe Cys Tyr Gly 50 55 60 Asn
Arg Val Phe Ala Lys Tyr Pro Glu Asn Ile Val Asp Tyr Phe Lys 65 70
75 80 Gln Ser Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met Asn Tyr
Glu 85 90 95 Asp Gly Gly Ile Cys Asn Ala Thr Asn Asp Ile Thr Leu
Asp Gly Asp 100 105 110 Cys Tyr Ile Tyr Glu Ile Arg Phe Asp Gly Val
Asn Phe Pro Ala Asn 115 120 125 Gly Pro Val Met Gln Lys Arg Thr Val
Lys Trp Glu Pro Ser Thr Glu 130 135 140 Lys Leu Tyr Val Arg Asp Gly
Val Leu Lys Gly Asp Val Asn Met Ala 145 150 155 160 Leu Ser Leu Glu
Gly Gly Gly His Tyr Arg Cys Asp Phe Lys Thr Thr 165 170 175 Tyr Lys
Ala Lys Lys Val Val Gln Leu Pro Asp Tyr His Phe Val Asp 180 185 190
His His Ile Glu Ile Lys Ser His Asp Lys Asp Tyr Ser Asn Val Asn 195
200 205 Leu His Glu His Ala Glu Ala His Ser Glu Leu Pro Arg Gln Ala
Lys 210 215 220 2672DNAArtificialDronpa-145K 2atgagcgtga tcaagcccga
catgaaaatc aagctgagga tggagggtgc cgtgaacggt 60cacccgtttg cgatcgaggg
cgtgggactg ggcaagccgt tcgagggaaa gcaaagcatg 120gacctcaagg
tgaaggaggg cggaccgctg cccttcgcct acgacattct gaccactgtc
180ttctgctacg gtaatcgcgt cttcgcaaag tatcccgaaa acatcgtgga
ttactttaag 240cagagcttcc cggagggtta ctcctgggag cgatccatga
actacgagga cggagggatc 300tgcaacgcga cgaacgatat aaccttggac
ggcgactgct acatttacga aattcggttc 360gacggcgtga atttcccagc
gaatggccct gtaatgcaaa aacgcacggt aaagtgggag 420cccagcaccg
agaagctgta cgtccgcgac ggagtcctga agggagacgt gaacatggcg
480ctttcattgg aaggtggcgg gcactacaga tgcgatttca agacgaccta
taaggcgaaa 540aaggtggtcc aactgccgga ctaccatttc gtcgaccacc
acatcgagat caagagccac 600gacaaagact atagcaacgt gaatctccac
gagcatgccg aggcccacag cgagctgccc 660aggcaggcca ag
6723224PRTArtificialDronpa-145N 3Met Ser Val Ile Lys Pro Asp Met
Lys Ile Lys Leu Arg Met Glu Gly 1 5 10 15 Ala Val Asn Gly His Pro
Phe Ala Ile Glu Gly Val Gly Leu Gly Lys 20 25 30 Pro Phe Glu Gly
Lys Gln Ser Met Asp Leu Lys Val Lys Glu Gly Gly 35 40 45 Pro Leu
Pro Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe Cys Tyr Gly 50 55 60
Asn Arg Val Phe Ala Lys Tyr Pro Glu Asn Ile Val Asp Tyr Phe Lys 65
70 75 80 Gln Ser Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met Asn
Tyr Glu 85 90 95 Asp Gly Gly Ile Cys Asn Ala Thr Asn Asp Ile Thr
Leu Asp Gly Asp 100 105 110 Cys Tyr Ile Tyr Glu Ile Arg Phe Asp Gly
Val Asn Phe Pro Ala Asn 115 120 125 Gly Pro Val Met Gln Lys Arg Thr
Val Lys Trp Glu Pro Ser Thr Glu 130 135 140 Asn Leu Tyr Val Arg Asp
Gly Val Leu Lys Gly Asp Val Asn Met Ala 145 150 155 160 Leu Ser Leu
Glu Gly Gly Gly His Tyr Arg Cys Asp Phe Lys Thr Thr 165 170 175 Tyr
Lys Ala Lys Lys Val Val Gln Leu Pro Asp Tyr His Phe Val Asp 180 185
190 His His Ile Glu Ile Lys Ser His Asp Lys Asp Tyr Ser Asn Val Asn
195 200 205 Leu His Glu His Ala Glu Ala His Ser Glu Leu Pro Arg Gln
Ala Lys 210 215 220 4672DNAArtificialDronpa-145N 4atgagcgtga
tcaagcccga catgaaaatc aagctgagga tggagggtgc cgtgaacggt 60cacccgtttg
cgatcgaggg cgtgggactg ggcaagccgt tcgagggaaa gcaaagcatg
120gacctcaagg tgaaggaggg cggaccgctg cccttcgcct acgacattct
gaccactgtc 180ttctgctacg gtaatcgcgt cttcgcaaag tatcccgaaa
acatcgtgga ttactttaag 240cagagcttcc cggagggtta ctcctgggag
cgatccatga actacgagga cggagggatc 300tgcaacgcga cgaacgatat
aaccttggac ggcgactgct acatttacga aattcggttc 360gacggcgtga
atttcccagc gaatggccct gtaatgcaaa aacgcacggt aaagtgggag
420cccagcaccg agaacctgta cgtccgcgac ggagtcctga agggagacgt
gaacatggcg 480ctttcattgg aaggtggcgg gcactacaga tgcgatttca
agacgaccta taaggcgaaa 540aaggtggtcc aactgccgga ctaccatttc
gtcgaccacc acatcgagat caagagccac 600gacaaagact atagcaacgt
gaatctccac gagcatgccg aggcccacag cgagctgccc 660aggcaggcca ag
6725224PRTArtificialPadron-145N 5Met Ser Val Ile Lys Pro Asp Met
Lys Ile Lys Leu Arg Met Glu Gly 1 5 10 15 Ala Val Asn Gly His Pro
Phe Ala Ile Glu Gly Val Gly Leu Gly Lys 20 25 30 Pro Phe Glu Gly
Lys Gln Ser Met Asp Leu Lys Val Lys Glu Gly Gly 35 40 45 Pro Leu
Pro Phe Ala Tyr Asp Ile Leu Thr Met Ala Phe Cys Tyr Gly 50 55 60
Asn Arg Val Phe Ala Lys Tyr Pro Glu Asn Ile Val Asp Tyr Phe Lys 65
70 75 80 Gln Ser Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met His
Tyr Glu 85 90 95 Asp Gly Gly Ser Cys Asn Ala Thr Asn Asp Ile Thr
Leu Asp Gly Asp 100 105 110 Cys Tyr Ile Tyr Glu Ile Arg Phe Asp Gly
Val Asn Phe Pro Ala Asn 115 120 125 Gly Pro Val Met Gln Lys Arg Thr
Val Lys Trp Glu Arg Ser Thr Glu 130 135 140 Asn Leu Tyr Val Arg Asp
Gly Val Leu Lys Ser Asp Gly Asn Tyr Ala 145 150 155 160 Leu Ser Leu
Glu Gly Gly Gly His Tyr Arg Cys Asp Phe Lys Thr Thr 165 170 175 Tyr
Lys Ala Lys Lys Val Val Gln Leu Pro Asp Tyr His Ser Val Asp 180 185
190 His His Ile Glu Ile Lys Ser His Asp Lys Asp Tyr Ser Asn Val Asn
195 200 205 Leu His Glu His Ala Glu Ala His Ser Glu Leu Pro Arg Gln
Ala Asn 210 215 220 6675DNAArtificialPadron-145N 6atgagtgtga
ttaaaccaga catgaagatc aagctgcgta tggaaggcgc tgtaaatgga 60cacccgttcg
cgattgaagg agttggcctt gggaagcctt tcgagggaaa acagagtatg
120gaccttaaag tcaaagaagg cggacctctg cctttcgcct atgacatctt
gacaatggcg 180ttctgttacg gcaacagggt attcgccaaa tacccagaaa
atatagtaga ctatttcaag 240cagtcgtttc ctgagggcta ctcttgggag
cgaagcatgc attacgaaga cgggggctca 300tgtaacgcga caaacgacat
aaccctggat ggtgactgtt atatctatga aattcgattt 360gatggcgtga
actttcctgc caatggtcca gttatgcaga agaggactgt gaaatgggag
420cggtccactg agaacttgta tgtgcgtgat ggagtgctga agtctgatgg
taactacgct 480ctgtcgcttg aaggaggtgg ccattaccga tgtgacttca
aaactactta taaagctaag 540aaggttgtcc agttgccaga ctatcactct
gtggaccacc acattgagat taaaagccac 600gacaaagatt acagtaatgt
taatctgcat gagcatgccg aagcgcattc tgagctgccg 660aggcaggcca actaa
6757233PRTArtificialrsTagRFP 7Met Ser Glu Leu Ile Lys Glu Asn Met
His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His
Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly
Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu
Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly
Ser Arg Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Trp 65 70
75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr
Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser
Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Leu Arg Gly
Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr
Leu Gly Trp Glu Ala Ala Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp
Gly Gly Leu Glu Gly Arg Gly Asp Met 145 150 155 160 Ala Leu Lys Leu
Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr
Arg Ser Lys Asn Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190
Tyr Phe Val Asp His Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195
200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp
Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230
8759DNAArtificialrsTagRFP 8atgagcgagc tgattaagga gaacatgcac
atgaagctgt acatggaggg caccgtgaac 60aaccaccact tcaagtgcac atccgagggc
gaaggcaagc cctacgaggg cacccagacc 120atgagaatca aggtggtcga
gggcggccct ctccccttcg ccttcgacat cctggctacc 180agcttcatgt
acggcagccg caccttcatc aaccacaccc agggcatccc cgacttctgg
240aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
agacgggggc 300gtgctgaccg ctacccagga caccagcctc caggacggct
gcctcatcta caacgtcaag 360ctcagagggg tgaacttccc atccaacggc
cctgtgatgc agaagaaaac actcggctgg 420gaggccgcca ccgagatgct
gtaccccgct gacggcggcc tggaaggcag aggggacatg 480gccctgaagc
tcgtgggcgg gggccacctg atctgcaact tgaagaccac atacagatcc
540aagaatcccg ctaagaacct caagatgccc ggcgtctact ttgtggacca
cagactggaa 600agaatcaagg aggccgacaa agagacctac gtcgagcagc
acgaggtggc tgtggccaga 660tactgcgacc tccctagcaa actggggcac
aagcttaatt aagaattcga agcttggctg 720ttttggcgga tgagagaaga
ttttcagcct gatacagat 7599236PRTArtificialmApple-162H-164A 9Met Val
Ser Lys Gly Glu Glu Asn Asn Met Ala Ile Ile Lys Glu Phe 1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe 20
25 30 Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln
Thr 35 40 45 Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe
Ala Trp Asp 50 55 60 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys
Val Tyr Ile Lys His 65 70 75 80 Pro Ala Asp Ile Pro Asp Tyr Phe Lys
Leu Ser Phe Pro Glu Gly Phe 85 90 95 Arg Trp Glu Arg Val Met Asn
Phe Glu Asp Gly Gly Ile Ile His Val 100 105 110 Asn Gln Asp Ser Ser
Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys 115 120 125 Leu Arg Gly
Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys 130 135 140 Thr
Met Gly Trp Glu Ala Ser Glu Glu Arg Met Tyr Pro Glu Asp Gly 145 150
155 160 Ala His Lys Ala Glu Ile Lys Lys Arg Leu Lys Leu Lys Asp Gly
Gly 165 170 175 His Tyr Ala Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys
Lys Pro Val 180 185 190 Gln Leu Pro Gly Ala Tyr Ile Val Asp Ile Lys
Leu Asp Ile Val Ser 195 200 205 His Asn Glu Asp Tyr Thr Ile Val Glu
Gln Tyr Glu Arg Ala Glu Gly 210 215 220 Arg His Ser Thr Gly Gly Met
Asp Glu Leu Tyr Lys 225 230 235 10708DNAArtificialmApple-162H-164A
10atggtgagca agggcgagga gaataacatg gccatcatca aggagttcat gcgcttcaag
60gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
120cgcccctacg aggcctttca gaccgctaag ctgaaggtga ccaagggtgg
ccccctgccc 180ttcgcctggg acatcctgtc ccctcagttc atgtacggct
ccaaggtcta cattaagcac 240ccagccgaca tccccgacta cttcaagctg
tccttccccg agggcttcag gtgggagcgc 300gtgatgaact tcgaggacgg
cggcattatt cacgttaacc aggactcctc cctgcaggac 360ggcgtgttca
tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
420atgcagaaga agaccatggg ctgggaggcc tccgaggagc ggatgtaccc
cgaggacggc 480gcccataagt atgagatcaa gaagaggctg aagctgaagg
acggcggcca ctacgccgcc 540gaggtcaaga ccacctacaa ggccaagaag
cccgtgcagc tgcccggcgc ctacatcgtc 600gacatcaagt tggacatcgt
gtcccacaac gaggactaca ccatcgtgga acagtacgaa 660cgcgccgagg
gccgccactc caccggcggc atggacgagc tgtacaag 708114PRTArtificiallinker
11Gly Gly Ser Gly 1 125PRTArtificiallinker 12Gly Gly Gly Gly Ser 1
5 134PRTArtificiallinker 13Ser Ala Gly Gly 1
145PRTArtificialnuclear localization signal 14Lys Lys Lys Arg Lys 1
5 1526PRTArtificialmitochondrial localization sequence 15Met 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
164PRTArtificialendoplasmic reticulum localization sequence 16Lys
Asp Glu Leu 1 174PRTArtificialplasma membrane localization sequence
17Cys Xaa Xaa Xaa 1 184PRTArtificialplasma membrane localization
sequence 18Cys Cys Xaa Xaa 1 1914PRTHomo sapiens 19Lys Lys Lys Lys
Lys Lys Ser Lys Thr Lys Cys Val Ile Met 1 5 10 2042DNAHomo sapiens
20aagaagaaaa agaagaagtc caagactaag tgcgtgatca tg 422120PRTHomo
sapiens 21Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys
Thr Lys 1 5 10 15 Cys Val Ile Met 20
* * * * *