U.S. patent application number 16/256973 was filed with the patent office on 2019-08-01 for novel monomeric yellow-green fluorescent protein from cephalochordate.
The applicant listed for this patent is ALLELE BIOTECHNOLOGY AND PHARMACEUTICALS, INC.. Invention is credited to Gerard G. LAMBERT, Yuhui NI, Nathan C. Shaner, Jiwu WANG.
Application Number | 20190233482 16/256973 |
Document ID | / |
Family ID | 49997971 |
Filed Date | 2019-08-01 |
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United States Patent
Application |
20190233482 |
Kind Code |
A1 |
Shaner; Nathan C. ; et
al. |
August 1, 2019 |
NOVEL MONOMERIC YELLOW-GREEN FLUORESCENT PROTEIN FROM
CEPHALOCHORDATE
Abstract
The present disclosure provides isolated nucleic acid sequences
encoding a monomeric green/yellow fluorescent proteins, and
fragments and derivatives thereof. Also provided is a method for
engineering the nucleic acid sequence, a vector comprising the
nucleic acid sequence, a host cell comprising the vector, and use
of the vector in a method for expressing the nucleic acid sequence.
The present invention further provides an isolated nucleic acid, or
mimetic or complement thereof, that hybridizes under stringent
conditions to the nucleic acid sequence. Additionally, the present
invention provides a monomeric green/yellow fluorescent protein
encoded by the nucleic acid sequence, as well as derivatives,
fragments, and homologues thereof. Also provided is an antibody
that specifically binds to the green/yellow fluorescent
protein.
Inventors: |
Shaner; Nathan C.; (San
Diego, CA) ; LAMBERT; Gerard G.; (San Diego, CA)
; NI; Yuhui; (San Diego, CA) ; WANG; Jiwu;
(La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALLELE BIOTECHNOLOGY AND PHARMACEUTICALS, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
49997971 |
Appl. No.: |
16/256973 |
Filed: |
January 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13950239 |
Jul 24, 2013 |
10221221 |
|
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16256973 |
|
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61675237 |
Jul 24, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/46 20130101;
G16B 5/00 20190201; G16B 15/00 20190201; C07K 14/43504
20130101 |
International
Class: |
C07K 14/46 20060101
C07K014/46; G16B 15/00 20060101 G16B015/00; C07K 14/435 20060101
C07K014/435; G16B 5/00 20060101 G16B005/00 |
Claims
1-13. (canceled)
14. An isolated antibody to a polypeptide having the sequence of
SEQ ID NO: 1.
15. An isolated antibody to a polypeptide having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 1,
wherein the protein comprises at least one mutation selected from
the group consisting of: F15I, R25Q, A45D, Q56H, F67Y, K79V, S100V,
F115A, I118K, V140R, T141S, M143K, L144T, D156K, T158S, S163N,
Q168R, V171A, N174T, I185Y, and F192Y.
16. An isolated antibody to a protein a having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 1,
wherein the protein comprises at least one mutation selected from
the group consisting of: I118K and N174T, at least one mutation
selected from the group consisting of: V140R, L144T, D156K, T158S,
Q168R, and F192Y, and at least one mutation selected from the group
consisting of: R25Q, A45D, S163N, F15I, Q56H, F67Y, K79V, S100V,
F115A, T141S, M143K, V171A, and I185Y.
17. The isolated antibody of any of claims 1-3, wherein the
antibody is a monoclonal antibody.
18. The isolated antibody of any of claims 1-3, wherein the
antibody is a polyclonal antibody.
19. The isolated antibody of any of claims 1-3, wherein the
antibody is a V.sub.HH protein.
20. A binding fragment of any of the isolated antibody of any of
claims 1-3.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of the priority of U.S.
provisional application Ser. No. 61/675,237, filed Jul. 24,
2012.
FIELD OF THE INVENTION
[0002] The present disclosure relates to novel monomeric
yellow-green fluorescent proteins, termed mNeonGreen, which are
derived/generated from a tetrameric fluorescent protein from the
cephalochordate Branchiostoma lanceolatum. These exemplary
monomeric versions of the proteins disclosed herein were designed
and generated by structure and docking algorithms using protein
engineering techniques and have no known close structural or
functional homologs. The exemplary mNeonGreen monomeric fluorescent
proteins described herein are among the brightest known in its
class and have exceptional utility as a biomarker and/or protein
fusion tag, and have shown great usefulness as a FRET acceptor for
the newest generation of cyan fluorescent proteins.
BACKGROUND
[0003] Since the initial cloning of Aequorea victoria green
fluorescent protein (avGFP) over 20 years ago, fluorescent proteins
have become staples of biological imaging. After an initial flurry
of activity leading to the development of avGFP variants in the
blue to yellow-green wavelength range [1], the bulk of subsequent
fluorescent protein research has focused on expanding the
fluorescent protein color palette into the red region and improving
the brightness and performance of these longer-wavelength variants
[2], along with more recent improvements to cyan variants of avGFP
(CFPs) [3,4]. Since green and yellow variants of the original avGFP
(GFPs and YFPs) perform reliably for many applications, not much
effort has been placed on developing novel fluorescent proteins in
the green region of the spectrum. However, there is still room for
improvement of green and yellow fluorescent proteins, both for
routine imaging as well as more advanced applications such as
Forster resonance energy transfer (FRET) [5].
SUMMARY OF THE INVENTION
[0004] In view of the above, the present disclosure provides novel
green/yellow fluorescent proteins derived by protein engineering
based on exemplary yellow fluorescent proteins from Branchiostoma
lanceolatum (LanYFP, Allele Biotechnology, San Diego, Calif.),
which exhibits an unusually high quantum yield (.about.0.95) and
extinction coefficient (.about.150,000 M-1cm-1). In certain
embodiments, size exclusion chromatography revealed that these
proteins are tetramers (FIG. 1).
[0005] In one aspect, the disclosure provides an exemplary
compositions comprising a monomeric NeonGreen (mNeonGreen) protein
having the sequence of SEQ ID NO: 1. In one embodiment, the
composition comprises a protein having a polypeptide with at least
95% homology to the sequence set forth in SEQ ID No 1. In one
embodiment, the composition comprises a protein having a
polypeptide with at least 97% homology to SEQ ID No 1.
[0006] In one aspect, the present disclosure provides methods for
monomerization of tetrameric LanYFP and compositions comprising
thereof. In certain embodiments, the methods comprise the use of
structure prediction algorithms coupled with modeling of the
tetramer interfaces to guide the monomerization of LanYFP. In one
embodiment, the structure prediction algorithm is based on
well-conserved beta-barrel structures in fluorescent proteins.
[0007] In one aspect, the present disclosure provides an isolated
nucleic acid sequence encoding a non-oligomerizing green/yellow
Fluorescent Protein (having specific characteristic emission
spectrum described below). In certain embodiments, the nucleic acid
sequence may be compatible with mammalian (e.g., human) or other
species' codon usage.
[0008] In another embodiment, the disclosure provides a nucleic
acid sequence encoding a polypeptide that has at least about 95%
homology with the polypeptide encoded by the nucleic acid of SEQ ID
NO: 2. In yet another embodiment, a nucleic acid present in other
than its natural environment, wherein said nucleic acid encodes a
green/yellow chromoprotein or fluorescent mutant thereof, and
wherein said nucleic acid has a sequence identity of at least about
90% with SEQ ID NO: 2.
[0009] In still another aspect, the present disclosure provides a
vector that includes a nucleic acid sequence encoding a
non-oligomerizing green/yellow Fluorescent Protein (FP). In one
embodiment, the vector is a plasmid, a viral vector, or a linear
form of DNA template. In another embodiment, the nucleic acid
sequence of the vector is cDNA. Also provided is a host cell
comprising the vector. The present invention further provides use
of the vector in a method for expressing the nucleic acid sequence
in mammalian cells, plant cells, yeast cells, bacterial cells, etc.
In one embodiment, the nucleic acid sequence is expressed as a
tandem genetic fusion to another protein.
[0010] In one embodiment, the novel protein may be a monomer or
dimer. In a further embodiment, the lanYFP-derived fluorescent
protein comprises at least one or more of the following mutations
F15I, R25Q, A45D, Q56H, F67Y, K79V, S100V, F115A, I118K, V140R,
T141S, M143K, L144T, D156K, T158S, S163N, Q168R, V171A, N174T,
I185Y, F192Y.
[0011] In yet another aspect, the present disclosure provides an
antibody that specifically binds to the FPs of the invention. In
one embodiment, the antibody is a polyclonal antibody; in another
embodiment, the antibody is a monoclonal antibody; in yet another
embodiment, the antibody is a VHH protein.
[0012] Additional variations, aspects and advantages of the present
invention will be apparent in view of the following descriptions.
It should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given for the purpose of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from these detailed depictions and descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Size exclusion chromatography of LanYFP (tetramer),
dLanYFP (dimer), and mNeonGreen (monomer). The exemplary proteins
were detected with 480 nm excitation and 530 nm emission, compared
to mCherry (monomer) detected with 540 nm excitation and 620 nm
emission. Each exemplary fluorescent protein was run simultaneously
with mCherry as a size standard with detection at both wavelengths;
mCherry consistently eluted with the same peak shape and retention
time. Curves are normalized to peak fluorescence.
[0014] FIG. 2. Diagram of I-TASSER and RosettaDock models of
wild-type LanYFP. (A) A/B dimer interface with targeted residues
Ile118 and Asn174 shown as sticks and (B) A/C dimer interface with
targeted residues Val140, Leu144, and Asp156 shown as sticks.
[0015] FIG. 3. Sequence alignment of LanYFP, dLanYFP, and
mNeonGreen. A/B interface mutations are shown in red, A/C interface
mutations in yellow, and modified termini in green.
[0016] FIG. 4. Exemplary normalized absorbance, excitation, and
emission spectra of mNeonGreen.
[0017] FIG. 5. Fluorescence imaging of mNeonGreen fusion vectors.
Exemplary C-terminal mNeonGreen fusion constructs (with respect to
the fluorescent protein); for each fusion, the linker amino acid
length is indicated after the name of the targeted organelle or
fusion partner: (A) mNeonGreen-Annexin (A4)-C-12 (human; plasma
membrane); (B) mNeonGreen-.beta.-actin-C-18 (human; actin
cytoskeleton); (C) mNeonGreen-.beta.-Catenin-C-20 (mouse; tight
junctions); (D) mNeonGreen-CAAX-C-5 (20-amino acid farnesylation
signal from c-Ha-Ras; plasma membrane); (E) mNeonGreen-CAF1-C-10
(mouse chromatin assembly factor); (F) mNeonGreen-Caveolinl-C-10
(human); (G) mNeonGreen-Endosomes-C-14 (human RhoB GTPase); (H)
mNeonGreen-Fascin-C-10 (human; actin bundling); (I)
mNeonGreen-Fibrillarin-C-7 (human; nucleoli); (J)
mNeonGreen-FilaminA-C-14 (human; cytoskeleton); (K) mNeonGreen-LAMP
1-C-20 (rat; lysosomal membrane glycoprotein 1; lysosomes); (L)
mNeonGreen-Clathrin-C-15 (human, light chain B); (M)
mNeonGreen-Myotilin-C-14 (human; actin filaments); (N)
mNeonGreen-PCNA-C-19 (human; replication foci); (O)
mNeonGreen-Plastin-C-10 (human; actin binding); (P)
mNeonGreen-Rab4a-C-7 (human; endosomes); (Q) mNeonGreen-LC3B-C-7
(rat light chain; autophagosomes); (R) mNeonGreen-Talin-C-18
(mouse; focal adhesions); (S) mNeonGreen-Tubulin-C-35 (human;
microtubules); (T) mNeonGreen-ZO 1-C-14 (human; tight junctions).
The cell line used for expression of C-terminal mNeonGreen
constructs was Madin-Darby canine kidney (MDCK; ATCC, CCL-34) cells
in panels C and T. HeLa CCL2 (ATCC) cells were used in the
remaining panels. Scale bars represent 10 m.
[0018] FIG. 6. Additional fluorescence imaging of mNeonGreen fusion
vectors. Exemplary N-terminal mNeonGreen fusion constructs (with
respect to the fluorescent protein); for each fusion, the linker
amino acid length is indicated after the name of the targeted
organelle or fusion partner: (A) mNeonGreen-.alpha.-Actinin-N-19
(human; non-muscle; actin and focal adhesions); (B)
mNeonGreen-Calnexin-N-14 (human; endoplasmic reticulum); (C)
mNeonGreen-C-Src-N-7 (chicken; plasma membrane); (D)
mNeonGreen-Cx43-N-7 (rat .alpha.-1 connexin 43; gap junctions); (E)
mNeonGreen-EB3-N-7 (human microtubule-associated protein; RP/EB
family); (F) mNeonGreen-Keratin-N-17 (human; intermediate
filaments; cytokeratin 18); (G) mNeonGreen-Lamin B1-N-18 (human;
nuclear envelope); (H) mNeonGreen-Lifeact-N-7 (yeast; actin); (I)
mNeonGreen-MANNII-N-10 (mouse mannosidase 2; endoplasmic
reticulum); (J) mNeonGreen-MyosinIIA-N-14 (mouse non muscle; actin
binding); (K) mNeonGreen-Nup50-N-10 (human; nuclear pore complex);
(L) mNeonGreen-PDHAl-N-10 (human pyruvate dehydrogenase;
mitochondria); (M) mNeonGreen-PMP-N-10 (human peroxisomal membrane
protein; peroxisomes); (N) mNeonGreen-MAPTau-N-10 (human
microtubule associated protein); (0) mNeonGreen-TFR-N-20 (human
transferrin receptor; plasma membrane); (P) mNeonGreen-TPX2-N-10
(human; microtubules); (Q) mNeonGreen-VASP-N-10 (mouse; focal
adhesions); (R) mNeonGreen-VE-Cadherin-N-10 (human; adhesion
junctions); (S) mNeonGreen-Vimentin-N-7 (human; intermediate
filaments); (T) mNeonGreen-Zyxin-N-6 (human; focal adhesions). The
cell line used for expression N-terminal mNeonGreen constructs was
human cervical adenocarcinoma cells (HeLa-S3; ATCC, CCL-2.2) in
panels F, K, P and S. HeLa CCL2 (ATCC) cells were used in the
remaining panels. Scale bars represent 10 m.
[0019] FIG. 7. Fluorescence imaging of mNeonGreen H2B fusion
vectors. (A)-(D) C-terminal mNeonGreen-H2B-C-10 (human) in HeLa S3
cells; (A) interphase; (B) prophase; (C) metaphase; (D) anaphase;
(E)-(H) N-terminal mNeonGreen-H2B-N-6 (human) in HeLa S3 cells; (E)
interphase; (F) prophase; (G) metaphase; (H) anaphase. Scale bars
represent 10 m.
[0020] FIG. 8. Sample FRET experiments. (A) Emission spectra and
fixed and live cell FRET images for (A) mTurquoise-mNeonGreen, (B)
mTurquoise-mVenus, and (C) mCerulean-mVenus. Fixed Cells. The first
column is the post-AP image of donor with 405 nm excitation
illustrating increase in donor image intensity after bleaching an
ROI. The second column is the post-AP image of acceptor with 515 nm
excitation illustrating the same region has been photobleached. The
third column is a ratiometric image of the donor to represent the
increase in intensity of the bleached region, which is
representative of FRET efficiency (see FRET Efficiency Bar below).
Live Cells. The first column is the pre-AP image of both the donor
and acceptor, with each taking up half of the total image. The
second column is the post-AP image of the donor and acceptor
showing the increase in intensity of the donor and that the
acceptor was photobleached. The third column is a ratiometric image
of the donor to represent the increase in intensity of the bleached
cell, which is representative of FRET efficiency (see FRET
Efficiency Bar below). FRET Efficiency Bar. Placed on the
ratiometric image of a cell with a bleached region (Fixed cells) or
an entire bleached cell (Live cells). Blue-green represents an
average FRET efficiency of .about.0 and Pink-white is approaching
1.
DETAILED DESCRIPTION OF THE INVENTION
[0021] When describing the present invention, all terms not defined
herein have their common meanings recognized in the art. To the
extent that the following description is of a specific embodiment
or a particular use of the invention, it is intended to be
illustrative only, and not limiting of the claimed invention. The
following description is intended to cover all alternatives,
modifications and equivalents that are included in the spirit and
scope of the invention.
[0022] As used herein, typical monomerization required a known or a
previously solved crystalline structures of the target fluorescent
protein or a very close homolog thereof as a starting template.
Certain monomeric fluorescent proteins have been generated without
the use of solved crystal structures, but these cases have relied
on relatively close homology of predicted tetramer interface
residues with fluorescent proteins whose structures were already
known [9,10] (for example, mAG was engineered from Azami-Green
based on .about.52% peptide sequence identity (.about.74% positive)
to DsRed over the full length of the protein). The cephalochordate
fluorescent proteins are evolutionarily distant from other known
fluorescent proteins [11], with LanYFP displaying <40% sequence
identity to fluorescent proteins from all other classes of
organism, and only 20% homology (.about.42% positive) to the
closest homolog which has been monomerized (Kusabira-Orange [12]).
No structure has yet been solved for a cephalochordate fluorescent
protein. One exemplary compositions derived from such method as
described herein was designated as monomeric NeonGreen (mNeonGreen)
SEQ ID NO: 1.
[0023] In another embodiment, the disclosure provides a nucleic
acid sequence encoding a polypeptide that has at least about 95%
homology with the polypeptide encoded by the nucleic acid of SEQ ID
NO: 2. It is well understood by those of skill in the art that in
many instances modifications in particular locations in the
polypeptide sequence may have no effect upon the properties of the
resultant polypeptide. Unlike the specific mutations described in
detail herein, other mutations provide polypeptides which have
properties essentially or substantially indistinguishable from
those of the specific polypeptides disclosed herein (U.S. Pat. No.
6,800,733, which is incorporated herein by reference). In the case
represented in U.S. Pat. No. 6,800,733, it was accepted that amino
acid sequence of "a modified form of an Aequorea wild-type GFP
polypeptide is at least 95% homologous to the amino acid sequence"
with a disclosed core protein sequence including key amino acids
specified is within the genus of disclosed protein molecules.
[0024] In yet another embodiment, a nucleic acid present in other
than its natural environment, wherein said nucleic acid encodes a
green/yellow chromoprotein or fluorescent mutant thereof, and
wherein said nucleic acid has a sequence identity of at least about
90% with SEQ ID NO: 2. Variations of nucleic acid sequences that
are at least about 90% homologues that encode a chromo- or
fluorescent protein were accepted as within the defined genus (U.S.
Pat. No. 7,166,444, which is incorporated herein by reference).
[0025] In one aspect of the current disclosure, the inventors
utilized the I-TASSER server [13] to generate a theoretical
tertiary structure for LanYFP. The top structure generated by
I-TASSER was then used to model the tetramer structure by aligning
with known structures for tetrameric fluorescent proteins,
including dsFP483 (a cyan fluorescent protein from Discosoma sp.
[14]), which was predicted by I-TASSER to be one of its closest
tetrameric structural relatives. Starting configurations of the A/B
and A/C dimers based on these structural alignments were then used
as input to the RosettaDock algorithm [15], and several of the
lowest energy configurations predicted by RosettaDock were used for
selection of side chains to target for monomerization (FIG. 2).
[0026] In another aspect, the wild-type LanYFP gene was modified by
appending the first and last 7 amino acids of EGFP to the N and C
termini of the coding sequence, respectively, as has become common
practice with new fluorescent proteins [7], as well as an
additional 4 amino acid linker sequence (DNMA) before the
N-terminus of the original protein coding sequence. This
arrangement tends to improve folding and localization of many
fusions for many fluorescent proteins. Next, the inventors planned
a strategy based on the reliable paradigm of first breaking the
weaker (hydrophilic) A/B interface, rescuing fluorescence of the
resulting dimer by directed evolution, then breaking the stronger
(hydrophobic) A/C interface of the resulting bright dimer, and
finally rescuing the fluorescence of the monomer. This strategy was
highly successful in generating a monomeric variant of LanYFP based
on our structural models.
[0027] The present disclosure provides a method that indicated in
order to break the A/B interface of LanYFP, mutations I118K, which
was predicted to introduce a positive charge in near the center of
the predicted interface, and N174T which was expected to improve
folding, were accordingly created by molecular biology methods. As
expected, these mutations gave rise to a variant with substantially
reduced fluorescence but which migrated as a dimer on a
non-denaturing SDS-PAGE gel. Additional rounds of directed
evolution led to a bright dimeric variant, designated dLanYFP, with
the additional mutations A45D, S163N, and V171A, whose optical
properties are nearly identical to those of the parental tetramer
(see Table 1). This exemplary mutant was selected as the starting
point for developing the monomer.
[0028] The disclosure also indicated that in order to break the
dLanYFP A/C interface, the mutation D156K was initially selected to
introduce a positive charge close to the center of the predicted
interface. This mutant was almost entirely non-fluorescent, but
could be rescued through several rounds of directed evolution, with
final mutations Q56H, F67Y, S100T, F115A, T141S, and T158S. The
resulting variant, however, appeared to migrate only partially as a
monomer on non-denaturing SDS-PAGE, and almost entirely as a dimer
in size exclusion chromatography. To introduce additional positive
charge at the A/C interface and eliminate hydrophobic interactions,
the inventors next constructed a library incorporating the
additional mutations V140K/R and L144S/T/N, which the models used
by the inventors predicted to form the remaining hydrophobic patch
of the A/C interface. From this library, several fluorescent (but
dimmer) clones were identified, all of which migrated entirely as
monomers by size exclusion chromatography. Several additional
rounds of directed evolution eventually generated a monomeric clone
whose extinction coefficient and quantum yield approach the values
of the original tetramer.
[0029] In one embodiment, The final mutant, designated mNeonGreen,
contains a total of 21 mutations relative to tetrameric LanYFP
(F15I, R25Q, A45D, Q56H, F67Y, K79V, S100V, F115A, I118K, V140R,
T141S, M143K, L144T, D156K, T158S, S163N, Q168R, V171A, N174T,
I185Y, F192Y), in addition to the appended EGFP-type termini. Based
on our models, these mutations are distributed over the A/B
interface (I118K and N174T), the A/C interface (V140R, L144T,
D156K, T158S, Q168R, and F192Y), additional external regions (R25Q,
A45D, and S163N), and internal to the beta-barrel (F15I, Q56H,
F67Y, K79V, S100V, F115A, T141S, M143K, V171A, and I185Y). A
sequence alignment of LanYFP, dLanYFP, and mNeonGreen can be found
in the FIG. 3. The monomeric status of the final clone was verified
by size exclusion chromatography (FIG. 2).
[0030] In one embodiment of the disclosure, mNeonGreen displays
sharp excitation and emission peaks (506 nm and 517 nm, FIG. 4 and
Table 1) somewhat blue-shifted relative to the original tetrameric
LanYFP, placing it roughly midway between typical GFP and YFP
wavelength classes. As such, it was imaged quite efficiently using
standard GFP bandpass or long pass filter sets, or separated from
CFP signals with YFP filter sets. mNeonGreen is also among the
brightest monomeric fluorescent proteins yet described. Its high
quantum yield and extinction coefficient (Table 1) make it between
1.5 and 3 times as bright as commonly used GFPs and YFPs. Its
photostability is slightly higher than that of mEGFP under
widefield illumination (see Table 1), but somewhat lower for laser
illumination (.about.40% of mEGFP), within a practical range for
imaging applications. Its fluorescence pKa of .about.5.7 is similar
to most modern GFPs and YFPs. mNeonGreen does not display any
measurable sensitivity to Cl- ions.
[0031] In one aspect of the invention, in order to determine the
performance of mNeonGreen as a fluorescent probe in live cell
imaging, fusion vectors were constructed to both the N and C
terminus of the fluorescent protein. All fusions localized as
expected and mNeonGreen exhibited character typical of monomeric
fluorescent proteins in "difficult" fusions, including histone H2B,
connexins 26 and 43, and .alpha.-tubulin (FIGS. 5, 6, and 7).
Fusions of mNeonGreen with signal peptides and targeting proteins
confirmed expected localization patterns in the cytoskeleton
(.beta.-actin, Lifeact, fascin, cortactin, plastin (fimbrin), MAP
Tau, light chain myosin, myosin IIA, EB3, TPX2 and myotilin),
intermediate filaments (keratin and vimentin), the Golgi complex
(sialyltransferase, gal-T and mannosidase II), the nuclear envelope
(lamin B1), nuclear pores (Nup50), nucleus (CAF 1), endoplasmic
reticulum (calnexin and calreticulin), the plasma membrane (annexin
A4, CAAX, transferrin receptor, and C-src) nucleoli (fibrillarin),
mitochondria (pyruvate dehydrogenase and TOMM20), endosomes (Rab4a,
Rab5a, and RhoB GTPase), autophagosomes (LC3), centromeres (CENPB),
tight junctions (.beta.-catenin, VE-cadherin, and ZOl1), DNA
replication foci (PCNA) lysosomes (LAMP1), auto peroxisomes
(peroxisomal membrane protein), various vesicles (clathrin and
caveolin), and focal adhesions (.alpha.-actinin, talin, focal
adhesion kinase, filamin A, VASP, paxillin, vinculin and zyxin).
All phases of mitosis were observed in fusions of human histone H2B
to either the N or C terminus of mNeonGreen (FIG. 7).
[0032] In one specific embodiment of the current invention, because
of its high extinction coefficient and quantum yield, it was
expected that mNeonGreen would be a good FRET acceptor for cyan
fluorescent proteins. Most notably in the tests of this property, a
direct fusion of mNeonGreen to mTurquoise produced higher FRET
efficiency than the same construct using mVenus as the acceptor
(50% versus 41% as measured by acceptor photobleaching; 55% versus
43% as measured by fluorescence lifetime imaging (FLIM), see, for
example, FIG. 8). Thus, in one embodiment of the invention,
mNeonGreen can be used as an excellent choice as FRET acceptor for
many applications.
[0033] The present disclosure demonstrates that precise structural
characterization is not required for successful monomerization of a
novel fluorescent protein. Though LanYFP has very low sequence
identity to any fluorescent protein whose structure has been
solved, modeling of its tetrameric configuration utilizing
structure prediction and protein-protein docking algorithms
provided sufficient information to identify the side chains to
target for monomerization.
[0034] In one embodiment, the resulting monomeric variant,
mNeonGreen, has optical properties which are superior to those of
the most commonly used green and yellow fluorescent proteins, and
shows especially good promise as a FRET acceptor.
[0035] In another embodiment, since it shares so little sequence
identity with other commonly used fluorescent proteins, mNeonGreen
are useful target for antibody development, and are amenable to
orthogonal co-IP experiments along with jellyfish and coral-derived
fluorescent proteins.
[0036] The disclosed computation-based approach to fluorescent
protein engineering should open up possibilities for monomerizing
many additional oligomeric fluorescent proteins with potentially
superior optical properties whose structures have not yet been
solved.
TABLE-US-00001 SEQ ID 1:
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLK
STKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTM
QFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCR
SKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQP
MYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID 2:
atggtgagcaagggcgaggaggataacatggcctctctcccagcgacaca
tgagttacacatctttggctccatcaacggtgtggactttgacatggtgg
gtcagggcaccggcaatccaaatgatggttatgaggagttaaacctgaag
tccaccaagggtgacctccagttctccccctggattctggtccctcatat
cgggtatggcttccatcagtacctgccctaccctgacgggatgtcgcctt
tccaggccgccatggtagatggctccggataccaagtccatcgcacaatg
cagtttgaagatggtgcctcccttactgttaactaccgctacacctacga
gggaagccacatcaaaggagaggcccaggtgaaggggactggtaccctgc
tgacggtcctgtgatgaccaactcgctgaccgctgcggactggtgcaggt
cgaagaagacttaccccaacgacaaaaccatcatcagtacctttaagtgg
agttacaccactggaaatggcaagcgctaccggagcactgcgcggaccac
ctacacctttgccaagccaatggcggctaactatctgaagaaccagccga
tgtacgtgttccgtaagacggagctcaagcactccaagaccgagctcaac
ttcaaggagtggcaaaaggcctttaccgatgtgatgggcatggacgagct gtacaagtaa
TABLE-US-00002 TABLE 1 Physical and optical characteristics of
Branchiostoma lanceolatum-derived fluorescent proteins described
here, and other green and yellow fluorescent proteins in common
usage. Brightness Ex Em (% Protein (nm) (nm) .epsilon..sup..dagger.
.phi..sup..dagger-dbl. EGFP) Photostability.sup. pKa.sup..sctn.
Oligomerization LanYFP 513 524 150,000 0.95 424 ND 3.5 tetramer
dLanYFP 513 524 125,000 0.90 335 ND ND dimer mNeonGreen 506 517
115,000 0.80 274 158 5.7 monomer mCitrine.sup.# 516 529 77,000 0.76
174 49 5.7 monomer mVenus.sup.# 515 528 92,200 0.57 156 15 6.0
monomer EYFP.sup.# 514 527 83,400 0.61 151 60 6.9 weak dimer
mEmerald.sup.# 487 509 57,500 0.68 116 101 6.0 monomer mEGFP.sup.#
488 507 56,000 0.60 100 150 6.0 monomer .sup.#Data as reported
previously.sup.2, 16; .sup..dagger.Molar extinction coefficient
(M.sup.-1cm.sup.-1) determined by alkali denaturation
method.sup.16; .sup..dagger-dbl.Fluorescence quantum yield (see
Methods); .sup. Half-time for photobleaching under widefield
illumination starting from 1000 photons/s emitted per fluorescent
protein chromophore, as measured in live cells (see Methods).sup.8,
17. ND = not determined.
EXAMPLES
Example 1--Modeling
[0037] The primary amino acid sequence of wild-type LanYFP
(blFP-Y3, GenBank accession ACA48232) was used as input to the
I-TASSER structure prediction server
[18](http://zhanglab.ccmb.med.umich.edu/I-TAS SER/) using default
parameters. The top-scoring structure returned by I-TASSER was
aligned to the published tetramer crystal structure of dsFP483 (PDB
ID 3CGL) [19] to create two dimer configurations, which were
designated "A/B" and "A/C" for the homologous DsRed dimer
configurations [20,21] PDB files containing these starting dimer
configurations were used as input to the RosettaDock server [22]
(http://rosettadock.graylab.jhu.edu/) using default parameters.
Several of the lowest energy configurations returned by the
RosettaDock server for each dimer interface were used to identify
the side chains most likely to be involved in critical dimer
interactions.
Example 2--Cloning, Protein Expression, and Purification
[0038] All fluorescent protein coding sequences were inserted
between BamHI and EcoRI sites in the constitutive expression vector
pNCS which encodes an N-terminal 6.times.His tag and linker.
Fluorescent proteins were expressed in E. coli strain NEBTurbo (New
England Biolabs) or Mach1 (Invitrogen) by growing cultures in
2.times.YT medium supplemented with ampicillin overnight at
37.degree. C. and shaking at 250 rpm. Fluorescent proteins were
purified by Ni2+-affinity chromatography as previously describedl.
Proteins were eluted in 50 mM Tris pH 7.5 or 50 mM sodium phosphate
buffer pH 7.5 containing 250 mM imidazole. For all further
characterization experiments, eluted fluorescent proteins were
buffer-exchanged using Amicon Ultra0.5 10 kD MWCO ultrafiltration
units (Millipore) into the same buffer without imidazole. Proteins
were found to be stable when stored at 4.degree. C. indefinitely or
when frozen at -20.degree. C. or -80.degree. C.
Example 3--Directed Evolution
[0039] Multiple rounds of directed evolution and screening were
performed as previously described [23,24], with a summary of
techniques following here. Screening of FP-expressing E. coli
colonies was done by eye using a blue LED lamp and longpass yellow
filter. For each round of directed evolution, one to three of the
brightest clones from the previous round of were used as the
template for construction of randomly mutagenized libraries using
the GeneMorph II kit (Agilent Technologies). Mutagenic PCR
conditions were chosen such that the library would contain an
average of 2 to 4 mutations per clone. 20 to 30 of the brightest
clones identified by random mutagenesis were sequenced, and any
clones containing mutations predicted to revert the oligomeric
state of the protein were rej ected. The remaining amino acid
positions identified by random mutagenesis were partially or fully
randomized by directed mutagenesis by overlap extension PCR using
degenerate primers, with library sizes typically between 500 and
25,000 unique clones. The brightest clones from directed
mutagenesis were sequenced, optically characterized, and evaluated
for their oligomeric state by size exclusion chromatography. Those
clones which possessed superior optical properties while
maintaining the desired oligomeric state were used as the input for
the next round of directed evolution.
Example 4--Optical Characterization
[0040] For spectroscopy measurements, all samples and buffers were
filtered or centrifuged immediately before use. Purified
fluorescent protein (FP) samples were diluted into 10 mM Tris, pH
7.4 buffer and fluorescein (F2-; Sigma, St. Louis, Mo.) was diluted
into 0.1M NaOH. Absorbance measurements were collected with a Cary
Bio 100 UV-Vis Spectrophotometer (Varian Inc., Walnut Creek,
Calif.). Fluorescence measurements were collected with a Cary
Eclipse Spectrophotometer (Varian Inc.). All measurements for
absorbance were immediately preceded with a measured baseline with
the appropriate blank buffer. Fluorescence pKa values were
determined by measuring fluorescence emission of heavily diluted
purified dialyzed fluorescent protein samples in 100 mM mixed
citrate-Tris-glycine buffer with pH ranging from 3 to 11.
Example 5. Size Exclusion Chromatography
[0041] Purified and dialyzed fluorescent protein samples were
diluted into 50 mM Tris-HCl pH 7.5, 100 mM NaCl and filtered
through 0.2 .mu.m filters immediately prior to injection into a
Shimadzu Nexera UHPLC equipped with a Waters BEH200 1.7 .mu.m
4.6.times.150 mm size exclusion column and 4.6.times.30 mm guard
column. Samples were run in the same buffer at a flow rate of 0.3
ml per minute for a total run time of 20 minutes. Fluorescence of
the eluted protein was detected with an RF-20Axs fluorescence
detector (Shimadzu) with 480 nm and 540 nm excitation and 530 nm
and 620 nm emission wavelengths. Each LanYFP or variant sample was
co-injected with mCherry [24], which had been purified under
identical conditions and which served as a monomeric size standard.
A control run of mCherry alone displayed no bleedthrough into the
yellow emission channel.
Example 6. Quantum Yield (.phi.) and Extinction Coefficient
(.epsilon.)
[0042] A relatively concentrated stock sample of FP or F2--was
prepared and its full absorbance spectrum was measured with 0.5 nm
step size. This was done in the same cuvette to be used for
fluorescence spectra measurement. Identically absorbing solutions
(target OD.ltoreq.0.05) were separately prepared in quadruplicate
for the FP and F2--(.phi.=0.925) [25] and their emission spectra
were measured with 488 nm excitation. The excitation and emission
bandwidths were 2.5 nm and 5 nm, respectively with 1.0 nm step
size. Emission was collected for 490-750 nm and the integrated
intensities for each sample were calculated using the fluorimeter's
software. The average integrated intensities and their associated
absorbance values were used to calculate quantum yields as
previously described [24].
[0043] Absorbance spectra of purified FP samples were measured in
quadruplicate (0.5 nm step size) and were used to determine the
mean peak absorbance value. A baseline absorbance spectrum was then
measured with buffer diluted 1:1 with 2M NaOH (1M final
concentration). A double-concentration sample was prepared in half
the cuvette volume, mixed 1:1 with 2M NaOH, and its absorbance was
immediately measured. Data was acquired for NaOH-denatured protein
between 430 and 460 nm (with a peak .about.447 nm) and a full
UV-Vis spectrum was measured for the last sample to ensure the
protein fully denatured. Extinction coefficients were determined as
described previously [26,27], assuming that the denatured
chromophore absorbed with an extinction coefficient equivalent to a
denatured avGFP chromophore (44,000 M-1 cm-1).
Example 7. Excitation and Emission
[0044] For excitation spectra, fluorescence emission was monitored
at 535 nm. For emission spectra, fluorescence excitation was 465 nm
with a 5.0 nm bandpass throughout.
Example 8. Photobleaching
[0045] Laser-scanning confocal and widefield microscopy
photobleaching experiments utilized fusions of the appropriate
fluorescent protein to human histone H2B to allow for localized
fluorescence in the nucleus. HeLa S3 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented
with 12.5% fetal bovine serum (FBS; HyClone). The cells were then
seeded onto 35 mm Delta T imaging dishes (Bioptechs) for live cell
imaging. Approximately 24 hours after being seeded, cells were then
transfected with 1 .mu.g of DNA using Effectene (Qiagen) and
maintained in a 5% CO2 incubator for at least 24 hours before
imaging.
[0046] Widefield photobleaching was performed on a Nikon TE2000
inverted microscope equipped with a Nikon Plan Fluorite 40.times.
dry objective (NA=0.85) and an X-Cite Exacte metal halide lamp
(Lumen Dynamics, Mississauga, Ont). Photobleaching was conducted
using a Brightline FITC-HYQ filter cube (Chroma, Bellows Falls,
Vt.) and a Newport 1918-C(Newport, Rochester, N.Y.) optical power
meter was used to verify that the illumination power at the
objective was 4.3 mW. Power moderation was achieved by using
neutral density filters contained within the lamp. With neutral
density (ND32) in place, a region containing 10-20 evenly bright
nuclei was located. The neutral density was then removed from the
light path and the region was photobleached continuously with a 65
ms exposure time for 15 minutes for a total of 4700 frames. Images
were collected with a QImaging Retiga EXi camera (Photometrics,
Tucson, Ariz.). Multiple regions were photobleached to ensure that
data for 30 nuclei could be averaged. The raw data was collected
using NIS-Elements software (Nikon) and then analyzed with Simple
PCI software (Hamamatsu, Hamamatsu City, Japan).
[0047] Confocal photobleaching measurements were collected on an
Olympus FV1000 confocal microscope with an Olympus PLAPO 40.times.
oil-immersion objective (NA=1.0). A 488 nm Argon-ion laser line
(Melles Griot, Albuquerque, N. Mex.) was confirmed to be attuned to
an output power of 1005 W at the objective with a FieldMax II-TO
power meter (Coherent, Santa Clara, Calif.). The microscope was set
to a zoom of 2.times., a pinhole size of 500 .mu.m, a
photomultiplier voltage of 450V, an offset of 8, and a scan time of
4 s/pixel. Emission was collected with detector slit settings of
505-605 nm. Utilizing an output of minimum laser power, a region of
evenly bright nuclei was located. The laser power was raised back
to 1005 W and each region was photobleached continuously for
.about.8 minutes for a total of 300 frames, with multiple regions
being bleached to ensure data for 30 nuclei. Raw data was collected
with the FluoView software (Olympus) and then analyzed with Simple
PCI software (Hamamatsu).
[0048] All photobleaching data were scaled to represent the
equivalent of an emission rate of 1000 photons/s per fluorescent
protein chromophore at time zero as previously described6, 11, a
condition which produces a half-time of 150s for EGFP.
Example 9. Acceptor Photobleaching FRET (AP FRET)
[0049] FRET constructs of mTurquoise-mNeonGreen, mTurquoise-mVenus
and mCerulean-mVenus all contained a 10 amino acid
linker-SGLRSPPVAT between FPs12. All acceptor photobleaching
measurements were performed on an Olympus FV1000 confocal
microscope with a UPLAPO 40.times. oil immersion objective
(NA=1.0). A 515 nm Ar-ion laser line was used with a 458/515
dichroic mirror to excite and photobleach the mNeonGreen or mVenus
in each FRET pair. Emission during acceptor photobleaching was
collected in one channel spanning 528-553 nm to ensure bleaching of
all fluorescence. For each of the FRET constructs, a 405 nm diode
laser line was used with a 405/488 dichroic for excitation of the
CFP with one emission channel spanning 450-485 nm. The detector
gain was set to 685 volts, the offset was set to 8 and the scan
speed was set to 8.0 is/pixel. Each experiment was performed with a
pinhole size of 500 .mu.m.
[0050] For FRET efficiency measurements in live cells, a full view
image of the donor was acquired before and after acceptor
photobleaching of the entire cell. A region of interest (ROI) was
drawn over identical areas of the cell in each image and the
average intensities of these regions were calculated using the
microscope's software. The following formula was used to calculate
the FRET efficiency of each construct: FE=1-(Average intensity
donor PreAP/Average intensity donor PostAP) [29]
[0051] For FRET efficiency measurements in fixed cells, a full view
image of the donor was acquired before and after acceptor
photobleaching. An ROI was drawn over an evenly bright part of the
cell and acceptor photobleached. The average intensities of these
regions were calculated using the microscope's software and the
above FE formula was again used to calculate FRET efficiency.
Example 10. Frequency Domain Fluorescence Lifetime Measurements
(FD-FLIM)
[0052] The fluorescence lifetime measurements were made using a ISS
ALBA FastFLIM system (ISS Inc., Champaign, Ill.) coupled to an
Olympus IX71 microscope equipped with a 60 .times./1.2 NA
water-immersion objective lens. A Pathology Devices (Pathology
Devices, Inc.) stage top environmental control system maintains
temperature at 36.degree. C. and CO2 at 5%. A 5 mW 448 nm diode
laser was modulated by the FastFLIM module of the ALBA system at
the fundamental frequency of 20 MHzl3. The modulated laser is
coupled to the ALBA scanning system, which is controlled by the
VistaVision software (ISS Inc., Champaign, Ill.). The fluorescence
signals emitted from the specimen are routed by a beam splitter
through the 530/43 nm (acceptor emission) and the 480/40 (donor
emission) band-pass emission filters. The signals are then detected
using two identical avalanche photodiodes (APD). The phase delays
and modulation ratios of the emission relative to the excitation
are measured at seven modulation frequencies (20, 40, 60, 80, 100,
120, 140 MHz) for each pixel of an image.
[0053] The system is calibrated with the 50 .mu.M Coumarin 6
dissolved in ethanol (lifetime 2.5 ns) to provide the software with
a reference standard to estimate the lifetime values from the
experimental data [30]. Additionally, a second reference standard,
10 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) dissolved in
phosphate buffer (PB) pH 7.8 (lifetime of 5.4 ns) is used to check
that the system is accurately reporting the fluorescence lifetime
of a known sample. The distribution of the lifetimes for all the
pixels in the image is determined using the phasor (polar) plot
method [31'32]. For live-cell imaging, transfected cells grown in
chambered coverglass (2 well, Thermo Scientific) were identified by
epifluorescence microscopy, and then imaged by FD-FLIM using the
448 nm laser line. The laser power was adjusted to achieve
approximately 100,000 counts per second in the donor emission
channel, and frame averaging was used to accumulate approximately
200 peak counts per pixel. The data were analyzed with the
VistaVision software (ISS Inc., Champaign, Ill.) using a region
average for each selected square region of interest (ROI, typically
1-2 .mu.m).
Example 11. Fusion Plasmid Construction
[0054] mNeonGreen fluorescent protein expression vectors were
constructed using C1 and N1 (Clontech-style) cloning vectors. The
mNeonGreen cDNA was amplified with a 5' primer encoding an AgeI
site and a 3' primer encoding either a BspEI (C1) or NotI (Ni) site
for generating cloning vectors to create C-terminal and N-terminal
fusions (with regards to the FP), respectively. Purified and
digested PCR products were ligated into similarly digested EGFP-C1
and EGFP-N1 cloning vector backbones. To obtain targeting fusion
vectors, the appropriate cloning vector and a previously assembled
EGFP or mEmerald fusion vector were digested, either sequentially
or doubly, with the appropriate enzymes and ligated together after
gel purification.
[0055] Thus, to prepare mNeonGreen C-terminal fusions (number of
linker amino acids in parenthesis), the following digests were
performed: annexin A4 (12), NheI and BspEI (Alen Piljic, EMBL,
Heidelberg, Germany; NM 001153.3); .beta.-actin (7), NheI and BglII
(human .beta.-actin cDNA source: Clontech, Mountain View, Calif.;
NM_001101.3); .beta.-catenin (20), XhoI and BamHI (mouse
.beta.-catenin cDNA source: Origene, Rockville, Md.;
NM_001165902.1); 20 amino acid farnesylation signal from c-Ha-Ras
(CAAX; 5), AgeI and BspEI (c-Ha-Ras cDNA source: Clontech, Mountain
View, Calif.; NM_001130442.1); CAF1 (10), AgeI and BspEI (mouse
chromatin assembly factor cDNA source: Akash Gunjan, Florida State
University; NM_013733.3); caveolin 1 (10), NheI and BglII (human
caveolin 1 cDNA source: Origene; NM_001753); endosomes (14), NheI
and BspEI (endosomes cDNA source: Clontech; NM_004040.2); fascin
(10), BspEI and BamHI (human fascin cDNA source: Origene;
NM_003088.2); fibrillarin (7), AgeI and BspEI (fibrillarin cDNA
source: Evrogen, Moscow, Russia; NM_001436.3); filamin A (14),
BspEI and HindIII (human filamin cDNA source: David Calderwood,
Yale University; NM_001456.3); human lysosomal membrane
glycoprotein 1 (20), BamHI and NotI (LAMP1; George Patterson, NIH,
Bethesda Md., U.S.A.; NM_012857.1); human light chain clathrin
(15), NheI and BglII (human clathrin light chain cDNA source:
George Patterson, NIH; NM_001834.2); human myotilin, AgeI and BspEI
(MYOT; Origene; NM_006790.1); PCNA (19), AgeI and BspEI
(proliferating cell nuclear antigen cDNA source: David Gilbert,
FSU; NM_002592.2); plastin (10), BspEI and XhoI (human plastin 1
(fimbrin) cDNA source: Origene; NM_002670.1); canine Rab4a, BglII
and BamHI (Rab4a cDNA source: Viki Allen, U. Manchester, UK;
NM_004578.2); LC3B (7), AgeI and BspEI (rat LC3B cDNA source: Jenny
M. Tam, Harvard University; U05784.1); talin (22) AgeI and BspEI
(mouse talin 1 cDNA source: Clare Waterman, NIH; NM_011602.5);
.alpha.-tubulin (18), NheI and BglII (human .alpha.-tubulin cDNA
source: Clontech, Mountain View, Calif.; NM_006082).
[0056] To prepare mNeonGreen N-terminal fusions (number of linker
amino acids in parenthesis), the following digests were performed:
human non-muscle .alpha.-actinin, EcoRI and NotI (cDNA source, Tom
Keller, Florida State University (FSU), Tallahassee, Fla., U.S.A.;
NM_001130005.1); human calnexin, AgeI and NotI (Origene;
NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA
source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1);
connexin-43 (7), BamHI and NotI (rat Cx43 cDNA source: Matthias
Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA
source: Lynne Cassimeris, Lehigh University; NM_012326.2); human
keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, Ala.,
U.S.A.; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin
B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7),
BamHI and NotI (Lifeact cDNA source: IDT, Coralville, Iowa); mouse
mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and
BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH;
NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA
source: Origene; NM_022410.2); human nucleoporin 50 kDa, BamHI and
NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate
dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene;
NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP
cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and
NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20),
BamHI and NotI (transferrin receptor cDNA source: George Patterson,
NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA
source: Patricia Wadsworth, University of Massachusetts, Amherst;
NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare
Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI
and NotI (human VE cadherin cDNA source: Origene, Rockville, Md.;
NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA
source: Robert Goldman, Northwestern University; NM_003380.3),
zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene,
Rockville, Md.; NM_003461). All DNA for transfection was prepared
using the Plasmid Maxi kit (QIAGEN, Valencia, Calif.). To ensure
proper localization, mNeonGreen fusion proteins were characterized
by transfection in HeLa (S3 or CCL2 line) or MDCK cells (ATCC;
Manassas, Va.) using Effectene (QIAGEN; Valencia, Calif.) and
.about.1 g vector. Transfected cells were grown on coverslips in
DMEM/F12, fixed after 48 hours, and mounted with Gelvatol.
Epifluorescence images were captured with a Nikon 80i microscope
using widefield illumination and a Chroma FITC filter set to
confirm proper localization.
Example 12. Microscopy
[0057] All filters for fluorescence screening and imaging were
purchased from Chroma Technology (Bellows Falls, Vt.), Omega
Filters (Brattleboro, Vt.) or Semrock, Inc. (Rochester, N.Y.). HeLa
epithelial (CCL-2, ATCC, Manassas, Va., U.S.A.) and grey fox lung
fibroblast (CCL-168, ATCC) cells were grown in a 50:50 mixture of
DMEM (Dulbecco's modified Eagle's medium) and Ham's F12 with 12.5%
Cosmic calf serum (Thermo Scientific; Logan, Utah) and transfected
with Effectene (QIAGEN). Imaging was performed in Delta-T culture
chambers (Bioptechs; Butler, Pa.) under a humidified atmosphere of
5% CO2 in air. Imaging in widefield mode was performed with a Nikon
(Melville, N.Y.) TE-2000 inverted microscope equipped with Omega
QuantaMax.TM. filters and a Photometrics (Tucson, Ariz.) Cascade II
camera or an Olympus IX71 inverted microscope equipped with Semrock
BrightLine.TM. filters and a Hamamatsu (Bridgewater, N.J.)
ImagEM.TM. camera. Laser-scanning confocal microscopy was conducted
using a Nikon C1Si and an Olympus FV1000, both equipped with
argon-ion (457 and 488 nm) and helium-neon or diode (543 and 561 nm
respectively) lasers and proprietary filter sets. Spinning disk
confocal microscopy was performed on an Olympus DSUIX81 equipped
with a Lumen 200 illuminator (Prior Scientific; Boston, Mass.), a
Hamamatsu 9100-13 EMCCD camera, Semrock filters and ten-position
filter wheels driven by a Lambda 10-3 controller (Sutter). In some
cases, cell cultures expressing fluorescent protein fusions were
fixed after imaging in 2% (w/v) paraformaldehyde (Electron
Microscopy Sciences; Hatfield, Pa.) and washed several times in PBS
containing 0.05 M glycine before mounting with a polyvinyl
alcohol-based medium.
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Sequence CWU 1
1
61236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala
Ser Leu Pro Ala Thr1 5 10 15His Glu Leu His Ile Phe Gly Ser Ile Asn
Gly Val Asp Phe Asp Met 20 25 30Val Gly Gln Gly Thr Gly Asn Pro Asn
Asp Gly Tyr Glu Glu Leu Asn 35 40 45Leu Lys Ser Thr Lys Gly Asp Leu
Gln Phe Ser Pro Trp Ile Leu Val 50 55 60Pro His Ile Gly Tyr Gly Phe
His Gln Tyr Leu Pro Tyr Pro Asp Gly65 70 75 80Met Ser Pro Phe Gln
Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val 85 90 95His Arg Thr Met
Gln Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr 100 105 110Arg Tyr
Thr Tyr Glu Gly Ser His Ile Lys Gly Glu Ala Gln Val Lys 115 120
125Gly Thr Gly Phe Pro Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr
130 135 140Ala Ala Asp Trp Cys Arg Ser Lys Lys Thr Tyr Pro Asn Asp
Lys Thr145 150 155 160Ile Ile Ser Thr Phe Lys Trp Ser Tyr Thr Thr
Gly Asn Gly Lys Arg 165 170 175Tyr Arg Ser Thr Ala Arg Thr Thr Tyr
Thr Phe Ala Lys Pro Met Ala 180 185 190Ala Asn Tyr Leu Lys Asn Gln
Pro Met Tyr Val Phe Arg Lys Thr Glu 195 200 205Leu Lys His Ser Lys
Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala 210 215 220Phe Thr Asp
Val Met Gly Met Asp Glu Leu Tyr Lys225 230 2352711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
2atggtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca tgagttacac
60atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac cggcaatcca
120aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
gttctccccc 180tggattctgg tccctcatat cgggtatggc ttccatcagt
acctgcccta ccctgacggg 240atgtcgcctt tccaggccgc catggtagat
ggctccggat accaagtcca tcgcacaatg 300cagtttgaag atggtgcctc
ccttactgtt aactaccgct acacctacga gggaagccac 360atcaaaggag
aggcccaggt gaaggggact ggtttccctg ctgacggtcc tgtgatgacc
420aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
cgacaaaacc 480atcatcagta cctttaagtg gagttacacc actggaaatg
gcaagcgcta ccggagcact 540gcgcggacca cctacacctt tgccaagcca
atggcggcta actatctgaa gaaccagccg 600atgtacgtgt tccgtaagac
ggagctcaag cactccaaga ccgagctcaa cttcaaggag 660tggcaaaagg
cctttaccga tgtgatgggc atggacgagc tgtacaagta a 71136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag 3His
His His His His His1 5410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4Ser Gly Leu Arg Ser Pro Pro
Val Ala Thr1 5 105219PRTBranchiostoma lanceolatum 5Met Ser Leu Pro
Ala Thr His Glu Leu His Ile Phe Gly Ser Phe Asn1 5 10 15Gly Val Asp
Phe Asp Met Val Gly Arg Gly Thr Gly Asn Pro Asn Asp 20 25 30Gly Tyr
Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Ala Leu Gln Phe 35 40 45Ser
Pro Trp Ile Leu Val Pro Gln Ile Gly Tyr Gly Phe His Gln Tyr 50 55
60Leu Pro Phe Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Lys Asp65
70 75 80Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly
Ala 85 90 95Ser Leu Thr Ser Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His
Ile Lys 100 105 110Gly Glu Phe Gln Val Ile Gly Thr Gly Phe Pro Ala
Asp Gly Pro Val 115 120 125Met Thr Asn Ser Leu Thr Ala Ala Asp Trp
Cys Val Thr Lys Met Leu 130 135 140Tyr Pro Asn Asp Lys Thr Ile Ile
Ser Thr Phe Asp Trp Thr Tyr Thr145 150 155 160Thr Gly Ser Gly Lys
Arg Tyr Gln Ser Thr Val Arg Thr Asn Tyr Thr 165 170 175Phe Ala Lys
Pro Met Ala Ala Asn Ile Leu Lys Asn Gln Pro Met Phe 180 185 190Val
Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn Phe 195 200
205Lys Glu Trp Gln Lys Ala Phe Thr Asp Val Met 210
2156236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala
Ser Leu Pro Ala Thr1 5 10 15His Glu Leu His Ile Phe Gly Ser Phe Asn
Gly Val Asp Phe Asp Met 20 25 30Val Gly Arg Gly Thr Gly Asn Pro Asn
Asp Gly Tyr Glu Glu Leu Asn 35 40 45Leu Lys Ser Thr Lys Gly Asp Leu
Gln Phe Ser Pro Trp Ile Leu Val 50 55 60Pro Gln Ile Gly Tyr Gly Phe
His Gln Tyr Leu Pro Phe Pro Asp Gly65 70 75 80Met Ser Pro Phe Gln
Ala Ala Met Lys Asp Gly Ser Gly Tyr Gln Val 85 90 95His Arg Thr Met
Gln Phe Glu Asp Gly Ala Ser Leu Thr Ser Asn Tyr 100 105 110Arg Tyr
Thr Tyr Glu Gly Ser His Ile Lys Gly Glu Phe Gln Val Lys 115 120
125Gly Thr Gly Phe Pro Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr
130 135 140Ala Ala Asp Trp Cys Val Thr Lys Met Leu Tyr Pro Asn Asp
Lys Thr145 150 155 160Ile Ile Ser Thr Phe Asp Trp Thr Tyr Thr Thr
Gly Asn Gly Lys Arg 165 170 175Tyr Gln Ser Thr Ala Arg Thr Thr Tyr
Thr Phe Ala Lys Pro Met Ala 180 185 190Ala Asn Ile Leu Lys Asn Gln
Pro Met Phe Val Phe Arg Lys Thr Glu 195 200 205Leu Lys His Ser Lys
Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala 210 215 220Phe Thr Asp
Val Met Gly Met Asp Glu Leu Tyr Lys225 230 235
* * * * *
References